Treatment of cancer using a CD123 chimeric antigen receptor

ABSTRACT

The invention provides compositions and methods for treating diseases associated with expression of CD123. The invention also relates to chimeric antigen receptor (CAR) specific to CD123, vectors encoding the same, and recombinant cells comprising the CD123 CAR. The invention also includes methods of administering a genetically modified cell expressing a CAR that comprises a CD123 binding domain.

RELATED APPLICATIONS

This application claims priority to PCT Application No.PCT/CN2014/084696, filed Aug. 19, 2014, and PCT Application No.PCT/CN2014/090508, filed Nov. 6, 2014. The entire contents of each ofthese applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 18, 2015, isnamed N2067-7064WO5_SL.txt and is 625,588 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the use of immune effectolls(e.g., T cells or NK cells) engineered to express a Chimeric AntigenReceptor (CAR) to treat a disease associated with expression of theCluster of Differentiation 123 protein (CD123).

BACKGROUND OF THE INVENTION

Most patients with acute myeloid leukemia (AML) are incurable usingstandard therapy (Mrozek et al, 2012, J Clin Oncol, 30:4515-23) andthose with relapsed or refractory AML (RR-AML) have a particularly poorprognosis (Kern et al, 2003, Blood 2003, 101:64-70; Wheatley et al,1999, Br J Haematol, 107:69-79).

Genetic engineering can impart to T cells specificity toward a target ofchoice. T cells can be transduced with genetic material encoding asingle chain variable fragment (scFv) of an antibody, in conjunctionwith a signaling molecule, thereby using the complementarity determiningregion (CDR) to recognize a cell surface antigen in a non-MHC restrictedmanner. These cells are termed chimeric antigen receptor (CAR) T cells.Preclinical and clinical attempts to target at least 20 differentsurface molecules in a variety of malignancies have shown some activityyet were often limited by poor persistence of the infused CAR T cellproduct (Sadelain et al, 2009, Curr Opin Immunol 2009, 21:215-23).Recent success with anti-CD19 redirected T cells in patients withadvanced CLL and ALL (Porter et al, 2011, N Engl J Med, 365:725-33;Kalos et al, 2011, Science Transl Med, 3:95ra73; Grupp and Kalos, 2013,N Engl J Med, 368:1509-18) demonstrated that these cells can eradicatemassive tumor burden after a single infusion with remission lasting upto 3 years to date, underscoring the dramatic potential of CAR T celltherapy. There have been few preclinical attempts to target AML inanimal models (Marin et al, 2010, Haematologica, 95:2144-52; Tettamantiet al, 2013, Br J Haematol, 161:389-401) although a recently publishedsmall clinical trial demonstrated that it is feasible to produce andinfuse T cells to patients with an aggressive malignancy (Ritchie et al,2013, Mol Ther, 21:2122-9). Besides the ability for the chimeric antigenreceptor on the genetically modified T cells to recognize and destroythe targeted cells, a successful therapeutic T cell therapy needs tohave the ability to proliferate and persist over time, and to furthermonitor for relapse. T cells may be variably efficacious due to anergy,suppression or exhaustion but skilled practitioners have limited controlover these features at this time. To be effective, CAR transformedpatient T cells need to persist and maintain the ability to proliferatein response to antigen. It has been shown that ALL patient T cellsperform can do this with CART19 comprising a murine scFv (see, e.g.,Grupp et al., NEJM 368:1509-1518 (2013).

SUMMARY OF THE INVENTION

In a first aspect, the invention features an isolated nucleic acidmolecule encoding a chimeric antigen receptor (CAR), wherein the CARcomprises a CD123 binding domain (e.g., a human or humanized CD123binding domain), a transmembrane domain, and an intracellular signalingdomain (e.g., an intracellular signaling domain comprising acostimulatory domain and/or a primary signaling domain). In oneembodiment, the CAR comprises a CD123 binding domain described herein(e.g., a human or humanized CD123 binding domain described herein), atransmembrane domain described herein, and an intracellular signalingdomain described herein (e.g., an intracellular signaling domaincomprising a costimulatory domain and/or a primary signaling domain).

In one embodiment, the encoded CD123 binding domain comprises one ormore (e.g., all three) light chain complementary determining region 1(LC CDR1), light chain complementary determining region 2 (LC CDR2), andlight chain complementary determining region 3 (LC CDR3) of a CD123binding domain described herein, and/or one or more (e.g., all three)heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) of a CD123 binding domaindescribed herein, e.g., a CD123 binding domain comprising one or more,e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. Inone embodiment, the encoded CD123 binding domain (e.g., a human orhumanized CD123 binding domain) comprises a light chain variable regiondescribed herein (e.g., in Table 2, 6 or 9) and/or a heavy chainvariable region described herein (e.g., in Table 2, 6 or 9). In oneembodiment, the encoded CD123 binding domain is a scFv comprising alight chain and a heavy chain of an amino acid sequence of Table 2, 6 or9. In an embodiment, the CD123 binding domain (e.g., an scFv) comprises:a light chain variable region comprising an amino acid sequence havingat least one, two or three modifications (e.g., substitutions, e.g.,conservative substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions, e.g., conservative substitutions) of an amino acidsequence of a light chain variable region provided in Table 2, 6 or 9,or a sequence with 95-99% identity with an amino acid sequence of Table2. 6 or 9; and/or a heavy chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of a heavy chain variableregion provided in Table 2, 6 or 9, or a sequence with 95-99% identityto an amino acid sequence of Table 2, 6 or 9.

In other embodiments, the encoded CD123 binding domain comprises a HCCDR1, a HC CDR2, and a HC CDR3 of any CD123 heavy chain binding domainamino acid sequences listed in Table 2, 6 or 9. In embodiments, the CD33binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD123 binding domain comprises a LC CDR1, a LC CDR2,and a LC CDR3 of any CD123 light chain binding domain amino acidsequences listed in Table 2, 6 or 9.

In some embodiments, the encoded CD123 binding domain comprises one, twoor all of LC CDR1, LC CDR2, and LC CDR3 of any CD123 light chain bindingdomain amino acid sequences listed in Table 2 or 9, and one, two or allof HC CDR1, HC CDR2, and HC CDR3 of any CD123 heavy chain binding domainamino acid sequences listed in Table 2, 6 or 9.

In one embodiment, the encoded CD123 binding domain comprises an aminoacid sequence selected from a group consisting of SEQ ID NO:157-160,184-215, 478, 480, 483, and 485. In an embodiment, the encoded CD123binding domain (e.g., an scFv) comprises an amino acid sequence havingat least one, two or three modifications (e.g., substitutions, e.g.,conservative substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions, e.g., conservative substitutions) of an amino acidsequence of 157-160, 184-215, 478, 480, 483, and 485, or a sequence with95-99% identity with an amino acid sequence of SEQ ID NO: 157-160,184-215, 478, 480, 483, and 485.

In another embodiment, the encoded CD123 binding domain comprises aheavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 216-219 or 243-274, or an aminoacid sequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of SEQ ID NO: 216-219 or 243-274, or a sequence with95-99% identity to SEQ ID NO: 216-219 or 243-274. In another embodiment,the encoded CD123 binding domain comprises a heavy chain variable regioncomprising an amino acid sequence corresponding to the heavy chainvariable region of SEQ ID NO:478, 480, 483, or 485, or an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of the corresponding portion of SEQ ID NO:478, 480, 483,or 485, or a sequence with 95-99% identity to the corresponding portionof SEQ ID NO:478, 480, 483, or 485.

In another embodiment, the encoded CD123 binding domain comprises alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 275-278 or 302-333, or an aminoacid sequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of SEQ ID NO: 275-278 or 302-333, or a sequence with95-99% identity to SEQ ID NO: 275-278 or 302-333. In another embodiment,the encoded CD123 binding domain comprises a light chain variable regioncomprising an amino acid sequence corresponding to the light chainvariable region of SEQ ID NO:478, 480, 483, or 485, or an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of the corresponding portion of SEQ ID NO:478, 480, 483,or 485, or a sequence with 95-99% identity to the corresponding portionof SEQ ID NO:478, 480, 483, or 485.

In one embodiment, the nucleic acid molecule encoding the scFv comprisesa nucleotide sequence selected from the group consisting of SEQ ID NO:479, 481, 482, 484, or a sequence with 95-99% identity thereof. In oneembodiment, the nucleic acid molecule comprises a nucleotide sequenceencoding the heavy chain variable region and/or the light chain variableregion, wherein said nucleotide sequence comprises a portion of anucleotide sequence selected from the group consisting of SEQ ID NO:479, 481, 482, and 484, or a sequence with 95-99% identity thereof,corresponding to the heavy chain variable region and/or the light chainvariable region. In one embodiment, the nucleic acid molecule comprisesa nucleotide sequence encoding the heavy chain variable region and/orthe light chain variable region, wherein the encoded amino acid sequenceis selected from the group consisting of SEQ ID NO:157-160, or asequence with 95-99% identity thereof. In one embodiment, the nucleicacid molecule encodes an scFv comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:184-215, or a sequence with95-99% identity thereof. In one embodiment, the nucleic acid moleculecomprises a sequence encoding the heavy chain variable region and/or thelight chain variable region, wherein the encoded amino acid sequence isselected from the group consisting of SEQ ID NO:184-215, or a sequencewith 95-99% identity thereof.

In one embodiment, the encoded CD123 binding domain includes a(Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4(SEQ ID NO:26). The light chain variable region and heavy chain variableregion of a scFv can be, e.g., in any of the following orientations:light chain variable region-linker-heavy chain variable region or heavychain variable region-linker-light chain variable region.

In one embodiment, the encoded CAR includes a transmembrane domain thatcomprises a transmembrane domain of a protein, e.g., a protein describedherein, e.g., selected from the group consisting of the alpha, beta orzeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 andCD154. In one embodiment, the encoded transmembrane domain comprises thesequence of SEQ ID NO: 6. In one embodiment, the encoded transmembranedomain comprises an amino acid sequence comprising at least one, two orthree modifications but not more than 20, 10 or 5 modifications of theamino acid sequence of SEQ ID NO:6, or a sequence with 95-99% identityto an amino acid sequence of SEQ ID NO:6. In one embodiment, the nucleicacid sequence encoding the transmembrane domain comprises a nucleotidesequence of SEQ ID NO: 17, or a sequence with 95-99% identity thereof.

In one embodiment, the encoded CD123 binding domain is connected to thetransmembrane domain by a hinge region, e.g., a hinge region describedherein. In one embodiment, the encoded hinge region comprises SEQ IDNO:2, or a sequence with 95-99% identity thereof. In one embodiment, thenucleic acid sequence encoding the hinge region comprises a nucleotidesequence of SEQ ID NO: 13, or a sequence with 95-99% identity thereof.

In one embodiment, the isolated nucleic acid molecule further comprisesa sequence encoding a costimulatory domain, e.g., a costimulatory domaindescribed herein. In embodiments, the intracellular signaling domaincomprises a costimulatory domain. In embodiments, the intracellularsignaling domain comprises a primary signaling domain. In embodiments,the intracellular signaling domain comprises a costimulatory domain anda primary signaling domain.

In one embodiment, the encoded costimulatory domain is a functionalsignaling domain from a protein, e.g., described herein, e.g., selectedfrom the group consisting of a MHC class I molecule, TNF receptorproteins, Immunoglobulin-like proteins, cytokine receptors, integrins,signaling lymphocytic activation molecules (SLAM proteins), activatingNK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

In one embodiment, the encoded costimulatory domain of 4-1BB comprisesthe amino acid sequence of SEQ ID NO:7. In one embodiment, the encodedcostimulatory domain comprises an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO:7, or a sequencewith 95-99% identity to the amino acid sequence of SEQ ID NO:7. In oneembodiment, the nucleic acid sequence encoding the costimulatory domaincomprises the nucleotide sequence of SEQ ID NO:18, or a sequence with95-99% identity thereof. In another embodiment, the encodedcostimulatory domain of CD28 comprises the amino acid sequence of SEQ IDNO:43. In one embodiment, the encoded costimulatory domain comprises anamino acid sequence having at least one, two or three modifications butnot more than 20, 10 or 5 modifications of an amino acid sequence of SEQID NO:43, or a sequence with 95-99% identity to an amino acid sequenceof SEQ ID NO:43. In one embodiment, the nucleic acid sequence encodingthe costimulatory domain of CD28 comprises the nucleotide sequence ofSEQ ID NO:44, or a sequence with 95-99% identity thereof. In anotherembodiment, the encoded costimulatory domain of CD27 comprises the aminoacid sequence of SEQ ID NO:8. In one embodiment, the encodedcostimulatory domain comprises an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO:8, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO:8. In oneembodiment, the nucleic acid sequence encoding the costimulatory domainof CD27 comprises the nucleotide sequence of SEQ ID NO:19, or a sequencewith 95-99% identity thereof. In another embodiment, the encodedcostimulatory domain of ICOS comprises the amino acid sequence of SEQ IDNO:45. In one embodiment, the encoded costimulatory domain of ICOScomprises an amino acid sequence having at least one, two or threemodifications but not more than 20, 10 or 5 modifications of an aminoacid sequence of SEQ ID NO:45, or a sequence with 95-99% identity to anamino acid sequence of SEQ ID NO:45. In one embodiment, the nucleic acidsequence encoding the costimulatory domain of ICOS comprises thenucleotide sequence of SEQ ID NO:46, or a sequence with 95-99% identitythereof.

In one embodiment, the isolated nucleic acid molecule further comprisesa sequence encoding an intracellular signaling domain, e.g., anintracellular signaling domain described herein.

In some embodiments, the encoded primary signaling domain comprises afunctional signaling domain of CD3 zeta. In embodiments, the functionalsignaling domain of CD3 zeta comprises the amino acid sequence of SEQ IDNO:9 (mutant CD3 zeta) or SEQ ID NO:10 (wild type human CD3 zeta), or asequence with 95-99% identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of 4-1BB and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of 4-1BB comprises the sequence of SEQ ID NO: 7 and/orthe CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises an amino acidsequence having at least one, two or three modifications but not morethan 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:7and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or asequence with 95-99% identity to an amino acid sequence of SEQ ID NO:7and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the encoded intracellular signaling domain comprises thesequence of SEQ ID NO:7 and the sequence of SEQ ID NO:9 or SEQ ID NO:10,wherein the sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain. In oneembodiment, the nucleic acid sequence encoding the intracellularsignaling domain comprises the nucleotide sequence of SEQ ID NO:18, or asequence with 95-99% identity thereof, and/or the CD3 zeta nucleotidesequence of SEQ ID NO:20 or SEQ ID NO:21, or a sequence with 95-99%identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of CD27 and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of CD27 comprises the amino acid sequence of SEQ IDNO:8 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modificationsbut not more than 20, 10 or 5 modifications of an amino acid sequence ofSEQ ID NO:8 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:8 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the encoded intracellular signaling domaincomprises the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:9 orSEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain. In one embodiment, the nucleic acid sequence encodingthe intracellular signaling domain of CD27 comprises the nucleotidesequence of SEQ ID NO:19, or a sequence with 95-99% identity thereof,and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21,or a sequence with 95-99% identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of CD28 and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of CD28 comprises the amino acid sequence of SEQ IDNO:43 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modificationsbut not more than 20, 10 or 5 modifications of an amino acid sequence ofSEQ ID NO:43 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:43 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the encoded intracellular signaling domaincomprises the sequence of SEQ ID NO:43 and the sequence of SEQ ID NO:9or SEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain. In one embodiment, the nucleic acid sequence encodingthe intracellular signaling domain of CD28 comprises the nucleotidesequence of SEQ ID NO:44, or a sequence with 95-99% identity thereof,and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21,or a sequence with 95-99% identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of ICOS and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of ICOS comprises the amino acid sequence of SEQ IDNO:45 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modificationsbut not more than 20, 10 or 5 modifications of an amino acid sequence ofSEQ ID NO:45 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:45 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the encoded intracellular signaling domaincomprises the sequence of SEQ ID NO:45 and the sequence of SEQ ID NO:9or SEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain. In one embodiment, the nucleic acid sequence encodingthe intracellular signaling domain of ICOS comprises the nucleotidesequence of SEQ ID NO:46, or a sequence with 95-99% identity thereof,and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21,or a sequence with 95-99% identity thereof.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence, e.g., aleader sequence described herein, e.g., the amino acid sequence of SEQID NO: 1; a CD123 binding domain described herein, e.g., a CD123 bindingdomain comprising a LC CDR1, a LC CDR2, a LC CDR3, a HC CDR1, a HC CDR2and a HC CDR3 described herein, e.g., a human or humanized CD123 bindingdomain described in Table 2, 6 or 9, or a sequence with 95-99% identifythereof; a hinge region described herein, e.g., a hinge regioncomprising the amino acid sequence of SEQ ID NO:2; a transmembranedomain described herein, e.g., a transmembrane domain comprising theamino acid sequence of SEQ ID NO: 6; and an intracellular signalingdomain, e.g., an intracellular signaling domain described herein. In oneembodiment, the encoded intracellular signaling domain comprises acostimulatory domain, e.g., a costimulatory domain described herein(e.g., a 4-1BB costimulatory domain comprising the amino acid sequenceof SEQ ID NO:7, or a CD27 costimulatory domain comprising the amino acidsequence of SEQ ID NO:8), and/or a primary signaling domain, e.g., aprimary signaling domain described herein (e.g., a CD3 zeta stimulatorydomain comprising a sequence of SEQ ID NO:9 or SEQ ID NO:10). In oneembodiment, the isolated nucleic acid molecule encoding the CARconstruct includes a leader sequence encoded by the nucleic acidsequence of SEQ ID NO:12, or a sequence with 95-99% identity thereto.

In one embodiment, the isolated nucleic acid molecule comprises (e.g.,consists of) a nucleic acid encoding a CAR amino acid sequence of SEQ IDNO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 125,SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ IDNO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134,SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ IDNO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143,SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ IDNO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152,SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, or SEQ ID NO: 156; or anamino acid having one, two or three modifications (e.g., substitutions)but not more than 30, 20 or 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of an amino acid sequence of SEQ ID NO: 98,SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 125, SEQ IDNO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130,SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ IDNO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139,SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ IDNO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148,SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ IDNO: 153, SEQ ID NO: 154, SEQ ID NO: 155, or SEQ ID NO: 156; or an aminoacid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to anamino acid sequence of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQID NO: 101, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO:137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO:146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:155, or SEQ ID NO: 156.

In one embodiment, the isolated nucleic acid molecule comprises (e.g.,consists of) a nucleic acid sequence of SEQ ID NO:39, SEQ ID NO:40, SEQID NO:41, SEQ ID NO:42, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97 or a nucleic acidsequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to anucleic acid sequence of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQID NO:42, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, or SEQ ID NO:97.

In one aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CD123 binding domain, wherein the CD123 bindingdomain comprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a CD123 binding domain described herein, and/or one or more(e.g., all three) heavy chain complementary determining region 1 (HCCDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a CD123binding domain described herein, e.g., a human or humanized CD123binding domain comprising one or more, e.g., all three, LC CDRs and oneor more, e.g., all three, HC CDRs.

In other embodiments, the encoded CD123 binding domain comprises a HCCDR1, a HC CDR2, and a HC CDR3 of any CD123 heavy chain binding domainamino acid sequence listed in Table 2, 6 or 9. In embodiments, the CD123binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD123 binding domain comprises a LC CDR1, a LC CDR2,and a LC CDR3 of any CD123 light chain binding domain amino acidsequence listed in Table 2, 6 or 9.

In some embodiments, the encoded CD123 binding domain comprises one, twoor all of LC CDR1, LC CDR2, and LC CDR3 of any CD123 light chain bindingdomain amino acid sequences listed in Table 2, 6 or 9, and one, two orall of HC CDR1, HC CDR2, and HC CDR3 of any CD123 heavy chain bindingdomain amino acid sequences listed in Table 2, 6 or 9.

In one embodiment, the encoded CD123 binding domain comprises a lightchain variable region described herein (e.g., in SEQ ID NO: 275-278 or302-333) and/or a heavy chain variable region described herein (e.g., inSEQ ID NO:216-219 or 243-274). In one embodiment, the encoded CD123binding domain is a scFv comprising a light chain and a heavy chain ofan amino acid sequence of in SEQ ID NO: 157-160, 184-215, 478, 480, 483or 485. In an embodiment, the CD123 binding domain (e.g., an scFv)comprises: a light chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions) of an amino acid sequenceof a light chain variable region provided in SEQ ID NO: 275-278 or302-333, or a sequence with 95-99% identity with an amino acid sequenceof SEQ ID NO: 275-278 or 302-333; and/or a heavy chain variable regioncomprising an amino acid sequence having at least one, two or threemodifications (e.g., substitutions, e.g., conservative substitutions)but not more than 30, 20 or 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of an amino acid sequence of a heavy chainvariable region provided in SEQ ID NO: 216-219 or 243-274, or a sequencewith 95-99% identity to an amino acid sequence in SEQ ID NO: 216-219 or243-274.

In one embodiment, the encoded CD123 binding domain comprises an aminoacid sequence selected from a group consisting of SEQ ID NO:157, SEQ IDNO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:184, SEQ ID NO:185, SEQID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190,SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ IDNO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204,SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ IDNO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQID NO:214, SEQ ID NO:215, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:483and SEQ ID NO:485, or a sequence with 95-99% identify thereof. In oneembodiment, the encoded CD123 binding domain is a scFv, and a lightchain variable region comprising an amino acid sequence describedherein, e.g., in Table 2, 6 or 9, is attached to a heavy chain variableregion comprising an amino acid sequence described herein, e.g., inTable 2, 6 or 9, via a linker, e.g., a linker described herein. In oneembodiment, the encoded CD123 binding domain includes a (Gly4-Ser)nlinker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQ ID NO: 26).The light chain variable region and heavy chain variable region of ascFv can be, e.g., in any of the following orientations: light chainvariable region-linker-heavy chain variable region or heavy chainvariable region-linker-light chain variable region.

Polypeptides

In another aspect, the invention pertains to an isolated polypeptidemolecule encoded by the nucleic acid molecule. In one embodiment, theisolated polypeptide molecule comprises a sequence selected from thegroup consisting of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ IDNO: 101, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ IDNO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137,SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ IDNO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146,SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ IDNO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155,and SEQ ID NO: 156, or an amino acid sequence having at least one, twoor three modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of any of the aforesaidsequences, or a sequence with 95-99% identity to any of the aforesaidsequences.

In another aspect, the invention pertains to an isolated chimericantigen receptor (CAR) molecule (e.g., polypeptide) comprising a CD123binding domain (e.g., a human or humanized antibody or antibody fragmentthat specifically binds to CD123), a transmembrane domain, and anintracellular signaling domain (e.g., an intracellular signaling domaincomprising a costimulatory domain and/or a primary signaling domain). Inone embodiment, the CAR comprises an antibody or antibody fragment whichincludes a CD123 binding domain described herein (e.g., a human orhumanized antibody or antibody fragment that specifically binds to CD123as described herein), a transmembrane domain described herein, and anintracellular signaling domain described herein (e.g., an intracellularsignaling domain comprising a costimulatory domain and/or a primarysignaling domain described herein).

In one embodiment, the CD123 binding domain comprises one or more (e.g.,all three) light chain complementary determining region 1 (LC CDR1),light chain complementary determining region 2 (LC CDR2), and lightchain complementary determining region 3 (LC CDR3) of a CD123 bindingdomain described herein, and/or one or more (e.g., all three) heavychain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) of a CD123 binding domaindescribed herein, e.g., a CD123 binding domain comprising one or more,e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. Inone embodiment, the CD123 binding domain comprises a light chainvariable region described herein (e.g., in Table 2, 6 or 9) and/or aheavy chain variable region described herein (e.g., in Table 2, 6 or 9).In one embodiment, the CD123 binding domain is a scFv comprising a lightchain and a heavy chain of an amino acid sequence listed in Table 2, 6or 9. In an embodiment, the CD123 binding domain (e.g., an scFv)comprises: a light chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of a light chain variableregion provided in Table 2, 6 or 9, or a sequence with 95-99% identitywith an amino acid sequence provided in Table 2, 6 or 9; and/or a heavychain variable region comprising an amino acid sequence having at leastone, two or three modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of a heavy chain variable region provided in Table 2, 6 or 9,or a sequence with 95-99% identity to an amino acid sequence provided inTable 2, 6 or 9.

In other embodiments, the CD123 binding domain comprises a HC CDR1, a HCCDR2, and a HC CDR3 of any CD123 heavy chain binding domain amino acidsequences provided in Table 2, 6 or 9. In embodiments, the CD123 bindingdomain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD123 binding domain comprises a LC CDR1, a LC CDR2,and a LC CDR3) of any CD123 light chain binding domain amino acidsequences listed in Table 2, 6 or 9.

In some embodiments, the CD123 binding domain comprises one, two or allof LC CDR1, LC CDR2, and LC CDR3 of any CD123 light chain binding domainamino acid sequences listed in Table 2, 6 or 9, and one, two or all ofHC CDR1, HC CDR2, and HC CDR3 of any CD123 heavy chain binding domainamino acid sequences listed in Table 2, 6 or 9.

In one embodiment, the CD123 binding domain comprises an amino acidsequence selected from a group consisting of SEQ ID NO:157, SEQ IDNO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:184, SEQ ID NO:185, SEQID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190,SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ IDNO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204,SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ IDNO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQID NO:214, SEQ ID NO:215, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:483and SEQ ID NO:485; or an amino acid sequence having at least one, two orthree modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) to any of the aforesaidsequences; or a sequence with 95-99% identity to any of the aforesaidsequences. In one embodiment, the CD123 binding domain is a scFv, and alight chain variable region comprising an amino acid sequence describedherein, e.g., in Table 2, 6 or 9, is attached to a heavy chain variableregion comprising an amino acid sequence described herein, e.g., inTable 2, 6 or 9, via a linker, e.g., a linker described herein. In oneembodiment, the CD123 binding domain includes a (Gly4-Ser)n linker,wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQ ID NO: 26). Thelight chain variable region and heavy chain variable region of a scFvcan be, e.g., in any of the following orientations: light chain variableregion-linker-heavy chain variable region or heavy chain variableregion-linker-light chain variable region.

In one embodiment, the isolated CAR molecule comprises a transmembranedomain of a protein, e.g., a protein described herein, e.g., selectedfrom the group consisting of the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, thetransmembrane domain comprises a sequence of SEQ ID NO: 6. In oneembodiment, the transmembrane domain comprises an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions,e.g., conservative substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions, e.g., conservative substitutions) ofan amino acid sequence of SEQ ID NO: 6, or a sequence with 95-99%identity to an amino acid sequence of SEQ ID NO: 6.

In one embodiment, the CD123 binding domain is connected to thetransmembrane domain by a hinge region, e.g., a hinge region describedherein. In one embodiment, the encoded hinge region comprises SEQ IDNO:2, or a sequence with 95-99% identity thereof.

In one embodiment, the isolated CAR molecule further comprises asequence encoding a costimulatory domain, e.g., a costimulatory domaindescribed herein.

In some embodiments, the intracellular signaling domain of the isolatedCAR molecule comprises a costimulatory domain. In embodiments, theintracellular signaling domain of the isolated CAR molecule comprises aprimary signaling domain. In embodiments, the intracellular signalingdomain of the isolated CAR molecule comprises a costimulatory domain anda primary signaling domain.

In one embodiment, the costimulatory domain comprises a functionalsignaling domain of a protein selected from the group consisting of aMHC class I molecule, TNF receptor proteins, Immunoglobulin-likeproteins, cytokine receptors, integrins, signaling lymphocyticactivation molecules (SLAM proteins), activating NK cell receptors,BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40,CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D,NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a,and a ligand that specifically binds with CD83.

In one embodiment, the costimulatory domain of 4-1BB comprises thesequence of SEQ ID NO:7. In one embodiment, the costimulatory domaincomprises an amino acid sequence having at least one, two or threemodifications (e.g., substitutions, e.g., conservative substitutions)but not more than 20, 10 or 5 modifications (e.g., substitutions, e.g.,conservative substitutions) of an amino acid sequence of SEQ ID NO:7, ora sequence with 95-99% identity to an amino acid sequence of SEQ IDNO:7. In another embodiment, the costimulatory domain of CD28 comprisesthe amino acid sequence of SEQ ID NO:43. In one embodiment, thecostimulatory domain comprises an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO:43, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO:43. Inanother embodiment, the costimulatory domain of CD27 comprises the aminoacid sequence of SEQ ID NO:8. In one embodiment, the costimulatorydomain comprises an amino acid sequence having at least one, two orthree modifications but not more than 20, 10 or 5 modifications of anamino acid sequence of SEQ ID NO:8, or a sequence with 95-99% identityto an amino acid sequence of SEQ ID NO:8. In another embodiment, thecostimulatory domain of ICOS comprises the amino acid sequence of SEQ IDNO:45. In one embodiment, the costimulatory domain comprises an aminoacid sequence having at least one, two or three modifications, but notmore than 20, 10 or 5 modifications of an amino acid sequence of SEQ IDNO:45, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:45.

In embodiments, the primary signaling domain comprises a functionalsignaling domain of CD3 zeta. In embodiments, the functional signalingdomain of CD3 zeta comprises SEQ ID NO:9 (mutant CD3 zeta) or SEQ IDNO:10 (wild type human CD3 zeta), or a sequence with 95-99% identitythereof.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of 4-1BB and/or a functional signalingdomain of CD3 zeta. In one embodiment, the intracellular signalingdomain comprises the amino acid sequence of SEQ ID NO: 7 and/or theamino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment,the intracellular signaling domain comprises an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions,e.g., conservative substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions, e.g., conservative substitutions) ofthe amino acid sequence of SEQ ID NO: 7 and/or the amino acid sequenceof SEQ ID NO:9 or SEQ ID NO:10, or a sequence with 95-99% identity tothe amino acid sequence of SEQ ID NO: 7 and/or the amino acid sequenceof SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the intracellularsignaling domain comprises the amino acid sequence of SEQ ID NO: 7and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, whereinthe sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of CD27 and/or a functional signaling domainof CD3 zeta. In one embodiment, the intracellular signaling domain ofCD27 comprises the amino acid sequence of SEQ ID NO: 8 and/or the CD3zeta amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises an amino acidsequence having at least one, two or three modifications but not morethan 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:8and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or asequence with 95-99% identity to an amino acid sequence of SEQ ID NO:8and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises the sequence ofSEQ ID NO:8 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein thesequences comprising the intracellular signaling domain are expressed inthe same frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of CD28 and/or a functional signaling domainof CD3 zeta. In one embodiment, the encoded intracellular signalingdomain of CD28 comprises the amino acid sequence of SEQ ID NO:43 and/orthe CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises an amino acidsequence having at least one, two or three modifications but not morethan 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:43and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or asequence with 95-99% identity to an amino acid sequence of SEQ ID NO:43and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises the sequence ofSEQ ID NO:43 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, whereinthe sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of ICOS and/or a functional signaling domainof CD3 zeta. In one embodiment, the intracellular signaling domain ofICOS comprises the amino acid sequence of SEQ ID NO:45 and/or the CD3zeta amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises an amino acidsequence having at least one, two or three modifications but not morethan 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:45and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or asequence with 95-99% identity to an amino acid sequence of SEQ ID NO:381and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the encoded intracellular signaling domain comprises thesequence of SEQ ID NO:45 and the sequence of SEQ ID NO:9 or SEQ IDNO:10, wherein the sequences comprising the intracellular signalingdomain are expressed in the same frame and as a single polypeptidechain.

In one embodiment, the isolated CAR molecule further comprises a leadersequence, e.g., a leader sequence described herein. In one embodiment,the leader sequence comprises the amino acid sequence of SEQ ID NO: 1,or a sequence with 95-99% identity to an amino acid sequence of SEQ IDNO:1.

In another aspect, the invention pertains to an isolated CAR moleculecomprising a leader sequence, e.g., a leader sequence described herein,e.g., a leader sequence of SEQ ID NO: 1, or having 95-99% identitythereof, a CD123 binding domain described herein, e.g., a CD123 bindingdomain comprising a LC CDR1, a LC CDR2, a LC CDR3, a HC CDR1, a HC CDR2and a HC CDR3 described herein, e.g., a CD123 binding domain describedin Table 2 or 6, or a sequence with 95-99% identify thereof, a hingeregion, e.g., a hinge region described herein, e.g., a hinge region ofSEQ ID NO:2, or having 95-99% identity thereof, a transmembrane domain,e.g., a transmembrane domain described herein, e.g., a transmembranedomain having a sequence of SEQ ID NO:6 or a sequence having 95-99%identity thereof, an intracellular signaling domain, e.g., anintracellular signaling domain described herein (e.g., an intracellularsignaling domain comprising a costimulatory domain and/or a primarysignaling domain). In one embodiment, the intracellular signaling domaincomprises a costimulatory domain, e.g., a costimulatory domain describedherein, e.g., a 4-1BB costimulatory domain having a sequence of SEQ IDNO:7, or having 95-99% identity thereof, and/or a primary signalingdomain, e.g., a primary signaling domain described herein, e.g., a CD3zeta stimulatory domain having a sequence of SEQ ID NO:9 or SEQ IDNO:10, or having 95-99% identity thereof. In one embodiment, theintracellular signaling domain comprises a costimulatory domain, e.g., acostimulatory domain described herein, e.g., a 4-1BB costimulatorydomain having a sequence of SEQ ID NO:7, and/or a primary signalingdomain, e.g., a primary signaling domain described herein, e.g., a CD3zeta stimulatory domain having a sequence of SEQ ID NO:9 or SEQ IDNO:10.

In one embodiment, the isolated CAR molecule comprises (e.g., consistsof) an amino acid sequence of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQID NO: 155, and SEQ ID NO: 156, or an amino acid sequence having atleast one, two, three, four, five, 10, 15, 20 or 30 modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 60,50 or 40 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 156, or an amino acidsequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an aminoacid sequence of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ IDNO: 101, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ IDNO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137,SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ IDNO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146,SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ IDNO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155,and SEQ ID NO: 156.

In one aspect, the invention pertains to a CD123 binding domaincomprising one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a CD123 binding domain described herein, and/or one or more(e.g., all three) heavy chain complementary determining region 1 (HCCDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a CD123binding domain described herein, e.g., a CD123 binding domain comprisingone or more, e.g., all three, LC CDRs and one or more, e.g., all three,HC CDRs (e.g., one, two or three HC CDRs according to Tables 3, 7, 10 or12; and/or one, two or three LC CDRs according to Tables 4, 8, 11 or13).

In other embodiments, the CD123 binding domain comprises a HC CDR1, a HCCDR2, and a HC CDR3 of any CD123 heavy chain binding domain amino acidsequences listed in Table 2, 6 or 9. In embodiments, the CD123 bindingdomain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD123 binding domain comprises a LC CDR1, a LC CDR2,and a LC CDR3) of any CD123 light chain binding domain amino acidsequences listed in Table 2, 6 or 9.

In some embodiments, the CD123 binding domain comprises one, two or allof LC CDR1, LC CDR2, and LC CDR3 of any CD123 light chain binding domainamino acid sequences listed in Table 2, 6 or 9, and one, two or all ofHC CDR1, HC CDR2, and HC CDR3 of any CD123 heavy chain binding domainamino acid sequences listed in Table 2, 6 or 9.

In one embodiment, the CD123 binding domain comprises a light chainvariable region described herein (e.g., in SEQ ID NO:275-278 or 302-333)and/or a heavy chain variable region described herein (e.g. in SEQ IDNO:216-219 or 243-274). In one embodiment, the CD123 binding domain is ascFv comprising a light chain and a heavy chain of an amino acidsequence of SEQ ID NO:157-160, 184-215, 478, 480, 483 or 485. In anembodiment, the CD123 binding domain (e.g., an scFv) comprises: a lightchain variable region comprising an amino acid sequence having at leastone, two or three modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of a light chain variable region provided, in SEQ ID NO:275-278 or 302-333 or a sequence with 95-99% identity with an amino acidsequence in SEQ ID NO: 275-278 or 302-333; and/or a heavy chain variableregion comprising an amino acid sequence having at least one, two orthree modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of a heavy chain variable region provided in SEQ ID NO: 216-219or 243-274, or a sequence with 95-99% identity to an amino acid sequencein SEQ ID NO: 216-219 or 243-274.

In one embodiment, the CD123 binding domain comprises a sequenceselected from a group consisting of SEQ ID NO:157, SEQ ID NO:158, SEQ IDNO:159, SEQ ID NO:160, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191,SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ IDNO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205,SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ IDNO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQID NO:215, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:483, and SEQ IDNO:485, or a sequence with 95-99% identify thereof. In one embodiment,the CD123 binding domain is a scFv, and a light chain variable regioncomprising an amino acid sequence described herein, e.g., in Table 2, 6or 9, is attached to a heavy chain variable region comprising an aminoacid sequence described herein, e.g., in Table 2, 6 or 9, via a linker,e.g., a linker described herein. In one embodiment, the CD123 bindingdomain includes a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6,preferably 4 (SEQ ID NO: 26). The light chain variable region and heavychain variable region of a scFv can be, e.g., in any of the followingorientations: light chain variable region-linker-heavy chain variableregion or heavy chain variable region-linker-light chain variableregion.

Nucleic Acids, Vectors and Cells

The nucleic acid molecules described herein can be a DNA molecule, anRNA molecule, or a combination thereof. In one embodiment, the nucleicacid molecule is an mRNA encoding a CAR polypeptide as described herein.In other embodiments, the nucleic acid molecule is a vector thatincludes any of the aforesaid nucleic acid molecules.

In another aspect, the invention pertains to a vector comprising anucleic acid molecule described herein, e.g., a nucleic acid moleculeencoding a CAR described herein. In one embodiment, the vector isselected from the group consisting of a DNA molecule or an RNA molecule(e.g., a plasmid, a lentivirus vector, adenoviral vector, or aretrovirus vector).

In one embodiment, the vector is a lentivirus vector. In one embodiment,the vector further comprises a promoter. In one embodiment, the promoteris an EF-1 promoter. In one embodiment, the EF-1 promoter comprises asequence of SEQ ID NO: 11. In another embodiment, the promoter is a PGKpromoter, e.g., a truncated PGK promoter as described herein.

In one embodiment, the vector is an in vitro transcribed vector, e.g., avector that transcribes RNA of a nucleic acid molecule described herein.In one embodiment, the nucleic acid sequence in the vector furthercomprises a poly(A) tail, e.g., a poly A tail described herein, (e.g.,comprising about 150 adenosine bases (SEQ ID NO: 705)). In oneembodiment, the nucleic acid sequence in the vector further comprises a3′UTR, e.g., a 3′ UTR described herein, e.g., comprising at least onerepeat of a 3′UTR derived from human beta-globulin. In one embodiment,the nucleic acid sequence in the vector further comprises promoter,e.g., a T2A promoter.

In another aspect, the invention pertains to a cell comprising a nucleicacid molecule or a vector, or expressing a CAR polypeptide as describedherein. In one embodiment, the cell is a cell described herein, e.g., animmune effector cell (e.g., a human T cell or NK cell, e.g., a human Tcell or NK cell as described herein, or a cell population thereof). Inone embodiment, the human T cell is a CD8+ T cell. In some embodiments,the cell expresses the CAR nucleic acid or polypeptide, or at some pointexpressed the CAR nucleic acid or polypeptide (e.g., a transientlyexpressed CAR molecule).

In some embodiment, the cell (e.g., the CAR-expressing cell) describedherein can further express another agent, e.g., an agent which enhancesthe activity of a CAR-expressing cell. For example, in one embodiment,the agent can be a chimeric molecule that comprises an inhibitorymolecule or a domain thereof. Examples of inhibitory molecules includePD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFRbeta), e.g.,as described herein. In one embodiment, the chimeric molecule comprisesa first polypeptide, e.g., an inhibitory molecule, associated with asecond polypeptide that provides a positive signal to the cell, e.g., anintracellular signaling domain described herein. In one embodiment, theagent comprises a first polypeptide, e.g., of an inhibitory moleculesuch as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80,CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,MHC class I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFRbeta),or a fragment of any of these (e.g., at least a portion of theextracellular domain of any of these), and a second polypeptide which isan intracellular signaling domain described herein (e.g., comprising acostimulatory domain (e.g., 41BB, CD27 or CD28, e.g., as describedherein) and/or a primary signaling domain (e.g., a CD3 zeta signalingdomain described herein). In one embodiment, the agent comprises a firstpolypeptide of PD1 or a fragment thereof (e.g., at least a portion ofthe extracellular domain of PD1), and a second polypeptide of anintracellular signaling domain described herein (e.g., a CD28 signalingdomain described herein and/or a CD3 zeta signaling domain describedherein).

In another aspect, the invention pertains to a method of making a cell,e.g., an immune effector cell. The method includes introducing into,e.g., transducing, the immune effector cell with a nucleic acid moleculedescribed herein (e.g., an RNA molecule, e.g., an mRNA), or a vectorcomprising a nucleic acid molecule encoding a CAR, e.g., a CAR describedherein.

The present invention also provides a method of generating a populationof cells (e.g., RNA-engineered cells transiently expressing an exogenousRNA). The method includes introducing into the cell an RNA as describedherein (e.g., an in vitro transcribed RNA or synthetic RNA; an mRNAsequence encoding a CAR polypeptide as described herein). Inembodiments, the RNA expresses the CAR polypeptide transiently. In oneembodiment, the cell is a cell as described herein, e.g., an immuneeffector cell (e.g., T cells or NK cells, or cell population).

Therapeutic Uses

In another aspect, the invention pertains to a method of providing ananti-tumor immunity in a mammal comprising administering to the mammalan effective amount of a cell expressing a CAR molecule, e.g., a cellexpressing a CAR molecule described herein. In one embodiment, the cellis an autologous immune effector cell, e.g., T cell or NK cell. In oneembodiment, the cell is an allogeneic immune effector cell, e.g., T cellor NK cell. In one embodiment, the mammal is a human, e.g., a patientwith a hematologic cancer.

In another aspect, the invention pertains to a method of treating amammal having a disease associated with expression of CD123 (e.g., aproliferative disease, a precancerous condition, or a non-cancer relatedindication associated with the expression of CD123). The method includesadministering to the mammal an effective amount of the cells expressinga CAR molecule, e.g., a CAR molecule described herein. In oneembodiment, the mammal is a human, e.g., a patient with a hematologiccancer.

In one embodiment, the disease is a disease described herein. In oneembodiment, the disease associated with CD123 expression is chosen from:a proliferative disease such as a cancer or a malignancy; a precancerouscondition such as a myelodysplasia, a myelodysplastic syndrome or apreleukemia; or a non-cancer related indication associated withexpression of CD123. In one embodiment, the disease is a hematologiccancer. In other embodiments, the disease is chosen from one or moreacute leukemias, including but not limited to, acute myeloid leukemia(AML), acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cellleukemia (B-cell acute lymphoid leukemia, BALL), and acute lymphoblasticT-cell leukemia (T-cell acute lymphoid leukemia (TALL); myelodysplasticsyndrome; a myeloproliferative neoplasm; a histiocytic disorder (e.g., amast cell disorder or a blastic plasmacytoid dendritic cell neoplasm); amast cell disorder, e.g., systemic mastocytosis or mast cell leukemia; achronic myeloid leukemia (CML); and a blastic plasmacytoid dendriticcell neoplasm. In other embodiments, the disease associated with CD123expression, includes, but is not limited to, atypical and/ornon-classical cancer, a malignancy, a precancerous condition or aproliferative disease expressing CD123; and a combination thereof.

In one embodiment, the disease is chosen from one or more of acutemyeloid leukemia (AML), acute lymphoblastic leukemia (ALL), acutelymphoblastic B-cell leukemia (B-cell acute lymphoid leukemia, BALL),acute lymphoblastic T-cell leukemia (T-cell acute lymphoid leukemia(TALL), B-cell prolymphocytic leukemia, chronic lymphocytic leukemia,chronic myeloid leukemia (CML), hairy cell leukemia, Hodgkin lymphoma, amast cell disorder, a histiocytic disorder, a myelodysplastic syndrome,a myeloproliferative neoplasm, a plasma cell myeloma, a blasticplasmacytoid dendritic cell neoplasm, or a combination thereof. In oneembodiment, the disease is a leukemia, e.g., ALL (e.g., relapsing andrefractory ALL) or AML. In other embodiments, the disease is aCD19-negative cancer, e.g., a CD19-negative relapsed cancer.

In some embodiments of any of the aforesaid methods, the cell, e.g., thepopulation of immune effector cells, comprises a vector, e.g., alentiviral vector, comprising a nucleic acid molecule encoding the CARpolypeptide as described herein.

In other embodiments of any of the aforesaid methods, the cell, e.g.,the population of immune effector cells, comprises an mRNA encoding theCAR polypeptide as described herein. In one embodiment, the cell is aCAR-expressing population of RNA-engineered cells, e.g., a population oftransiently expressing cells.

In some embodiments of any of the aforesaid methods, the method furtherincludes administering one or more doses of a cell (e.g., an immune cellcontaining a CAR nucleic acid or CAR polypeptide as described herein),to a mammal (e.g., a mammal having a cancer, e.g., a hematologic canceras described herein (e.g., AML or ALL)). In some embodiments, the one ormore doses of CAR cells (e.g., CD123 CAR cells) comprises at least about1×10⁶, 5×10⁶, 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or5×10⁹ cells.

In one embodiment, up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 doses of cellsare administered. In other embodiments, one, two, three, four, five or 6doses of the cells are administered to the mammal, e.g., in a treatmentinterval of one, two, three, four or more weeks. In one embodiment, upto 6 doses are administered in two weeks. The doses may the same ordifferent. In one embodiment, a lower dose is administered initially,followed by one or more higher doses. In one exemplary embodiment, thelower dose is about 1×10⁵ to 1×10⁹ cells/kg, or 1×10⁶ to 1×10⁸ cells/kg;and the higher dose is about 2×10⁵ to 2×10⁹ cells/kg or 2×10⁶ to 2×10⁸cells/kg, followed by 3-6 doses of about 4×10⁵ to 4×10⁹ cells/kg, or4×10⁶ to 4×10⁸ cells/kg.

In one embodiment, the one or more doses of the cells are administeredafter one or more lymphodepleting therapies, e.g., a lymphodepletingchemotherapy. In one embodiment, the lymphodepleting therapy includes achemotherapy (e.g., cyclophosphamide).

In one embodiment, the one or more doses is followed by a celltransplant, e.g., an allogeneic hematopoietic stem cell transplant. Forexample, the allogeneic hematopoietic stem cell transplant occursbetween about 20 to about 35 days, e.g., between about 23 and 33 days.

In some embodiments, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with one or more therapeutic agents orprocedures as described herein.

In one embodiment, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with an agent that increases the efficacy ofa cell expressing a CAR molecule, e.g., an agent described herein.

In one embodiment, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with a low, immune enhancing dose of an mTORinhibitor. While not wishing to be bound by theory, it is believed thattreatment with a low, immune enhancing, dose (e.g., a dose that isinsufficient to completely suppress the immune system but sufficient toimprove immune function) is accompanied by a decrease in PD-1 positive Tcells or an increase in PD-1 negative cells. PD-1 positive T cells, butnot PD-1 negative T cells, can be exhausted by engagement with cellswhich express a PD-1 ligand, e.g., PD-L1 or PD-L2.

In an embodiment this approach can be used to optimize the performanceof CAR cells described herein in the subject. While not wishing to bebound by theory, it is believed that, in an embodiment, the performanceof endogenous, non-modified immune effector cells, e.g., T cells, isimproved. While not wishing to be bound by theory, it is believed that,in an embodiment, the performance of a CD123 CAR expressing cell isimproved. In other embodiments, cells, e.g., T cells, which have, orwill be engineered to express a CAR, can be treated ex vivo by contactwith an amount of an mTOR inhibitor that increases the number of PD1negative immune effector cells (e.g., T cells or NK cells), or increasesthe ratio of PD1 negative immune effector cells, e.g., T cells or NKcells/PD1 positive immune effector cells, e.g., T cells or NK cells.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or acatalytic inhibitor, is initiated prior to administration of an CARexpressing cell described herein, e.g., T cells or NK cells. In anembodiment, the CAR cells are administered after a sufficient time, orsufficient dosing, of an mTOR inhibitor, such that the level of PD1negative immune effector cells, e.g., T cells, or the ratio of PD1negative immune effector cells, e.g., T cells or NK cells/PD1 positiveimmune effector cells, e.g., T cells or NK cells, has been, at leasttransiently, increased.

In an embodiment, the cell, e.g., immune effector cell (e.g., T cell orNK cell), to be engineered to express a CAR, is harvested after asufficient time, or after sufficient dosing of the low, immuneenhancing, dose of an mTOR inhibitor, such that the level of PD1negative immune effector cells, e.g., T cells or NK cells, or the ratioof PD1 negative immune effector cells, e.g., T cells or NK cells/PD1positive immune effector cells, e.g., T cells or NK cells, in thesubject or harvested from the subject has been, at least transiently,increased.

In an embodiment, the invention provides an mTOR inhibitor for use inthe treatment of a subject, wherein said mTOR inhibitor enhances animmune response of said subject, and wherein said subject has received,is receiving or is about to receive an immune effector cell thatexpresses a CD123 CAR as described herein.

In another embodiment, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with an agent that ameliorates one or moreside effect associated with administration of a cell expressing a CARmolecule, e.g., an agent described herein.

In one embodiment, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein), isadministered in combination with an agent that treats the diseaseassociated with CD123, e.g., an agent described herein.

In one embodiment, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with a second therapeutic agent or procedurechosen from one or more of chemotherapy, a targeted anti-cancer therapy,an oncolytic drug, a cytotoxic agent, a cytokine, surgical procedure, aradiation procedure, an agonist of a costimulatory molecule, aninhibitor of an immune checkpoint molecule, a vaccine, or a secondCAR-based immunotherapy.

In one embodiment, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with an agonist of a costimulatory molecule,e.g., an agonist of a costimulatory molecule chosen from one or more ofa MHC class I molecule, TNF receptor proteins, Immunoglobulin-likeproteins, cytokine receptors, integrins, signaling lymphocyticactivation molecules (SLAM proteins), activating NK cell receptors,BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40,CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D,NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a,and a ligand that specifically binds with CD83.

In other embodiments, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with an inhibitor of an immune checkpointmolecule chosen from one or more of PD1, PD-L1, PD-L2, CTLA4, TIM3,CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9,adenosine, TGFR (e.g., TGFR beta), or a combination thereof.

In one embodiment, the inhibitor of the immune checkpoint molecule orthe agonist of the costimulatory molecule is an antibody molecule, e.g.,a monospecific antibody molecule or a bispecific antibody molecule. Forexample, the cell, e.g., the population of immune effector cells, can beadministered in combination with a PD-1 inhibitor, a TIM-3 inhibitor, aCEACAM-1 inhibitor, or a combination thereof. In one embodiment, thePD-1 inhibitor and the TIM-3 inhibitor are administered in combination.In other embodiments, the TIM-3 inhibitor and the CEACAM-1 inhibitor areadministered in combination.

In some embodiments, the inhibitor of the immune checkpoint molecule isadministered subsequent to the administration of the cell, e.g., thepopulation of immune effector cells, e.g., about 3-7 days after theadministration of the cell, e.g., the population of immune effectorcells.

As described in the Examples herein, CAR123-expressing immune effectorcells have been shown to be an effective treatment for CD19-negativerelapses (e.g., CD19-negative B-ALL). Without being bound by theory, acombinatorial approach of CAR123- and CD19 inhibition (e.g.,CAR19-expressing immune effector cells) can be used to treatCD19-positive disease, while retarding or preventing antigen-lossrelapses.

Accordingly, in yet other embodiments of any of the aforesaid methods,the cell, e.g., the population of immune effector cells, is administeredin combination with a CD19 inhibitor, e.g., a CD19 inhibitor asdescribed herein.

In some embodiments, the CD19 inhibitor is a CD19 CAR-expressing cell oran anti-CD19 antibody molecule. In one embodiment, the disease is aleukemia, e.g., ALL (e.g., relapsing and refractory ALL). In otherembodiments the disease is AML. In other embodiments, the disease is aCD19-negative cancer, e.g., a CD19-negative relapsed cancer.

Alternatively or in combination with any of the aforesaid methods, amethod of preventing a CD19-negative relapse in a mammal, e.g., a human,is provided. The method includes administering an effective amount of acell, e.g., a population of immune effector cells (e.g., cellsexpressing a CAR molecule described herein). In some embodiments, themethod further includes administering a CD19 inhibitor, e.g., a CD19CAR-expressing cell. In some embodiments, the disease is a leukemia,e.g., acute lymphoblastic leukemia (e.g., relapsing and refractory ALL),or AML.

In some embodiments, the cell, e.g., the population of immune effectorcells, is administered before, simultaneously or concurrently, or afteradministration of the CD19 inhibitor. In one embodiment, the cell, e.g.,the population of immune effector cells, is administered afteradministration of the CD19. In other embodiments, the cell, e.g., thepopulation of immune effector cells, is administered concurrently withthe CD19 inhibitor.

In some embodiments, the cell, e.g., the population of immune effectorcells, expresses a CD19 CAR and the CD123 CAR (e.g., a CAR as describedherein).

Without wishing to be bound by theory, certain leukemic cells (e.g.,B-ALL basts) have been found to co-express CD19 and CD123, whilehemapoietic stem cells (HSC) are typically CD123-positive (CD123+), butCD19-negative (CD19−). In order to target leukemic cells that co-expressCD19 and CD123 (e.g., B-ALL basts) while sparing HSC, a split CAR can beused where the functional intracellular domains are separated betweenCAR19 and CAR123. Such CAR molecules would cause full activation of theimmune cell preferentially when the target cells co-express CD19 andCD123 (e.g., B-ALL basts), as opposed to a cell that expresses one ofCD19 or CD123 (e.g. HSCs).

Accordingly, in some embodiments, the CD19 CAR or CD123 CAR comprises asplit intracellular signaling domain such that full activation of thecell, e.g., the population of immune effector cells, occurs when boththe CD19 CAR and CD123 CAR bind to a target cell, e.g., a targetCD19+CD123+ cell (e.g., a B-ALL blast cell), compared to activation whenthe CD19 CAR and CD123 CAR bind to a target cell that expresses one ofCD19 or CD123 (e.g., a hematopoietic stem cell). In one embodiment, theCD123CAR comprises a costimulatory domain, e.g., 4-1BB signaling domain,and the CD19 CAR comprises a primary signaling domain, e.g., a CD3 zetasignaling domain. In other embodiments, the CD123CAR comprises acomprises a primary signaling domain, e.g., a CD3 zeta signaling domain,and the CD19 CAR comprises a costimulatory domain, e.g., 4-1BB signalingdomain. The CD123CAR and the CD19 CAR may be optionally linked to, e.g.,one or more peptide cleavage sites such as P2A.

In yet other embodiments, the methods disclosed herein further includeadministering a T cell depleting agent after treatment with the cell(e.g., an immune effector cell as described herein), thereby reducing(e.g., depleting) the CAR-expressing cells (e.g., theCD123CAR-expressing cells). Such T cell depleting agents can be used toeffectively deplete CAR-expressing cells (e.g., CD123CAR-expressingcells) to mitigate toxicity.

For example, alternatively or in combination with the methods disclosedherein, a method of reducing (e.g., depleting) a CAR-expressing cellafter a CAR therapy (e.g., a CAR123 therapy disclosed herein) isdisclosed. The method includes administering to a mammal a T celldepleting agent, in an amount to reduce (e.g., deplete) theCAR-expressing cells. In some embodiments, the T cell depleting agent isadministered after treatment of the mammal with a cell, e.g., apopulation of immune effector cells (e.g., a CAR-expressing populationof cells), thereby reducing (e.g., depleting) the cell (e.g., theCAR-expressing cell).

In some embodiments, the method further includes transplanting a cell,e.g., a hematopoietic stem cell, or a bone marrow, into the mammal.

In some embodiments, the mammal has a leukemia, e.g., acutelymphoblastic leukemia.

In some embodiments, the T cell depleting agent is administered one,two, three, four, or five weeks after administration of the cell, e.g.,the population of immune effector cells, described herein.

In one embodiment, the T cell depleting agent is an agent that depletesCAR-expressing cells, e.g., by inducing antibody dependent cell-mediatedcytotoxicity (ADCC) and/or complement-induced cell death. For example,CAR-expressing cells described herein may also express an antigen (e.g.,a target antigen) that is recognized by molecules capable of inducingcell death, e.g., ADCC or complement-induced cell death. For example,CAR expressing cells described herein may also express a target protein(e.g., a receptor) capable of being targeted by an antibody or antibodyfragment. Examples of such target proteins include, but are not limitedto, EpCAM, VEGFR, integrins (e.g., integrins ανβ3, α4, αI¾β3, α4β7,α5β1, ανβ3, αν), members of the TNF receptor superfamily (e.g.,TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor, folatereceptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a/LFA-1, CD15,CD18/ITGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25, CD28, CD30, CD33,CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125,CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7, andEGFR, and truncated versions thereof (e.g., versions preserving one ormore extracellular epitopes but lacking one or more regions within thecytoplasmic domain).

In some embodiments, the CAR expressing cell co-expresses the CAR andthe target protein, e.g., naturally expresses the target protein or isengineered to express the target protein. For example, the cell, e.g.,the population of immune effector cells, can include a nucleic acid(e.g., vector) comprising the CAR nucleic acid (e.g., a CAR nucleic acidas described herein) and a nucleic acid encoding the target protein.

In one embodiment, the T cell depleting agent is a CD52 inhibitor, e.g.,an anti-CD52 antibody molecule, e.g., alemtuzumab.

In other embodiments, the cell, e.g., the population of immune effectorcells, expresses a CAR molecule as described herein (e.g., CD123CAR) andthe target protein recognized by the T cell depleting agent. In oneembodiment, the target protein is CD20. In embodiments where the targetprotein is CD20, the T cell depleting agent is an anti-CD20 antibody,e.g., rituximab.

In further embodiments of any of the aforesaid methods, the methodsfurther include transplanting a cell, e.g., a hematopoietic stem cell,or a bone marrow, into the mammal.

In another aspect, the invention features a method of conditioning amammal prior to cell transplantation. The method includes administeringto the mammal an effective amount of the cell comprising the CAR nucleicacid as described herein, or the polypeptide as described herein. Insome embodiments, the cell transplantation is a stem celltransplantation, e.g., a hematopoietic stem cell transplantation, or abone marrow transplantation. In other embodiments, conditioning asubject prior to cell transplantation includes reducing the number ofCD123-expressing cells in a subject, e.g., CD123-expressing normal cellsor CD123-expressing cancer cells.

In another aspect, the invention pertains to the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use as amedicament, e.g., as described herein (e.g., for use in the treatment ofa disease associated with expression of CD123).

In another aspect, the invention pertains to the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use in thetreatment of a disease expressing CD123, e.g., a disease expressingCD123 as described herein. In certain embodiments, the disease is ahematologic cancer, e.g., as described herein. In some embodiments, thedisease is chosen from: an acute leukemia, including but not limited to,acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), acutelymphoblastic B-cell leukemia (B-cell acute lymphoid leukemia, BALL),and acute lymphoblastic T-cell leukemia (T-cell acute lymphoid leukemia(TALL); myelodysplastic syndrome; a myeloproliferative neoplasm; ahistiocytic disorder (e.g., a mast cell disorder or a blasticplasmacytoid dendritic cell neoplasm); a mast cell disorder, e.g.,systemic mastocytosis or mast cell leukemia; a chronic myeloid leukemia(CML); or a blastic plasmacytoid dendritic cell neoplasm.

Additional features and embodiments of the aforesaid compositions andmethods include one or more of the following:

In certain embodiments, the CD123 CAR molecule (e.g., a CD123 CARnucleic acid or a CD123 CAR polypeptide as described herein), or theCD123 binding domain as described herein, includes one, two or threeCDRs from the heavy chain variable region (e.g., HC CDR1, HC CDR2 and/orHC CDR3), provided in Table 3; and/or one, two or three CDRs from thelight chain variable region (e.g., LC CDR1, LC CDR2 and/or LC CDR3)provided in Table 4 (e.g., one, two or three HC CDR1, HC CDR2 or HCCDR3, and/or one, two or three LC CDR1, LC CDR2 or LC CDR3, of CAR123-1,CAR123-2, CAR123-3, CAR123-4, provided in Table 3 or 4); or a sequencesubstantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or1 amino acid changes, e.g., substitutions (e.g., conservativesubstitutions)) to any of the aforesaid sequences.

In certain embodiments, the CD123 CAR molecule (e.g., a CD123 CARnucleic acid or a CD123 CAR polypeptide as described herein), or theanti-CD123 antigen binding domain as described herein, includes one, twoor three CDRs from the heavy chain variable region (e.g., HC CDR1, HCCDR2 and/or HC CDR3), provided in Table 10; and/or one, two or threeCDRs from the light chain variable region provided in Table 11 (e.g.,one, two or three HC CDR1, HC CDR2 or HC CDR3, and/or one, two or threeLC CDR1, LC CDR2 or LC CDR3, of CAR123-1, CAR123-2, CAR123-3, CAR123-4,provided in Table 10 or 11); or a sequence substantially identical(e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid changes,e.g., substitutions (e.g., conservative substitutions)) to any of theaforesaid sequences.

In certain embodiments, the CD123 CAR molecule, or the anti-CD123antigen binding domain, includes one, two or three CDRs from the heavychain variable region (e.g., HCDR1, HCDR2 and/or HCDR3), provided inTable 12; and/or one, two or three CDRs from the light chain variableregion provided in Table 13 (e.g., one, two or three HC CDR1, HC CDR2 orHC CDR3, and/or one, two or three LC CDR1, LC CDR2 or LC CDR3, ofCAR123-1, CAR123-2, CAR123-3, CAR123-4, provided in Table 12 or 13); ora sequence substantially identical (e.g., 95-99% identical, or up to 5,4, 3, 2, or 1 amino acid changes, e.g., substitutions (e.g.,conservative substitutions)) to any of the aforesaid sequences.

In certain embodiments, the CD123 CAR molecule, or the anti-CD123antigen binding domain, includes

(i) a LC CDR1, LC CDR2 and LC CDR3 of any CD123 light chain bindingdomain amino acid sequences listed in Table 2, 6 or 9, or the LC CDRs inTable 4, 8, 11 or 13; and/or.

(ii) a HC CDR1, HC CDR2 and HC CDR3 of any CD123 heavy chain bindingdomain amino acid sequences listed in Table 2, 6 or 9, or the HC CDRs inTable 3, 7, 10 or 12.

In certain embodiments, the CD123 CAR molecule (e.g., a CD123 CARnucleic acid or a CD123 CAR polypeptide as described herein), or theanti-CD123 antigen binding domain as described herein, includes:

-   (1) one, two or three light chain (LC) CDRs chosen from one of the    following:

(i) a LC CDR1 of SEQ ID NO:418, LC CDR2 of SEQ ID NO:446 and LC CDR3 ofSEQ ID NO:474 of CAR123-1;

(ii) a LC CDR1 of SEQ ID NO:419, LC CDR2 of SEQ ID NO:447 and LC CDR3 ofSEQ ID NO:475 of CAR123-2;

(iii) a LC CDR1 of SEQ ID NO:420, LC CDR2 of SEQ ID NO:448 and LC CDR3of SEQ ID NO:476 of CAR123-3;

(iv) a LC CDR1 of SEQ ID NO:421, LC CDR2 of SEQ ID NO:449 and LC CDR3 ofSEQ ID NO:477 of CAR123-4; and/or

-   (2) one, two or three heavy chain (HC) CDRs chosen from one of the    following:

(i) a HC CDR1 of SEQ ID NO:334, HC CDR2 of SEQ ID NO:362 and HC CDR3 ofSEQ ID NO: 390 of CAR123-1;

(ii) a HC CDR1 of SEQ ID NO: 335, HC CDR2 of SEQ ID NO: 363 and HC CDR3of SEQ ID NO: 391 of CAR123-2;

(iii) a HC CDR1 of SEQ ID NO: 336, HC CDR2 of SEQ ID NO: 364 and HC CDR3of SEQ ID NO: 392 of CAR123-3;

(iv) a HC CDR1 of SEQ ID NO: 337, HC CDR2 of SEQ ID NO: 365 and HC CDR3of SEQ ID NO: 393 of CAR123-4.

In certain embodiments, the CD123 CAR molecule (e.g., a CD123 CARnucleic acid or a CD123 CAR polypeptide as described herein), or theanti-CD123 antigen binding domain as described herein, includes:

-   (1) one, two or three light chain (LC) CDRs chosen from one of the    following:

(i) a LC CDR1 of SEQ ID NO:501, LC CDR2 of SEQ ID NO: 506 and LC CDR3 ofSEQ ID NO: 511 of CAR123-1;

(ii) a LC CDR1 of SEQ ID NO: 502, LC CDR2 of SEQ ID NO: 507 and LC CDR3of SEQ ID NO: 512 of CAR123-2;

(iii) a LC CDR1 of SEQ ID NO: 503, LC CDR2 of SEQ ID NO: 508 and LC CDR3of SEQ ID NO: 513 of CAR123-3;

(iv) a LC CDR1 of SEQ ID NO: 504, LC CDR2 of SEQ ID NO: 509 and LC CDR3of SEQ ID NO: 514 of CAR123-4; and/or

-   (2) one, two or three heavy chain (HC) CDRs chosen from one of the    following:

(i) a HC CDR1 of SEQ ID NO:486, HC CDR2 of SEQ ID NO:491 and HC CDR3 ofSEQ ID NO: 496 of CAR123-1;

(ii) a HC CDR1 of SEQ ID NO: 487, HC CDR2 of SEQ ID NO:492 and HC CDR3of SEQ ID NO: 497 of CAR123-2;

(iii) a HC CDR1 of SEQ ID NO: 488, HC CDR2 of SEQ ID NO:493 and HC CDR3of SEQ ID NO: 498 of CAR123-3;

(iv) a HC CDR1 of SEQ ID NO: 489, HC CDR2 of SEQ ID NO:494 and HC CDR3of SEQ ID NO: 499 of CAR123-4.

In certain embodiments, the CD123 CAR molecule (e.g., a CD123 CARnucleic acid or a CD123 CAR polypeptide as described herein), or theanti-CD123 antigen binding domain as described herein, includes:

-   (1) one, two or three light chain (LC) CDRs chosen from one of the    following:

(i) a LC CDR1 of SEQ ID NO:531, LC CDR2 of SEQ ID NO: 536 and LC CDR3 ofSEQ ID NO: 541 of CAR123-1;

(ii) a LC CDR1 of SEQ ID NO: 532, LC CDR2 of SEQ ID NO: 537 and LC CDR3of SEQ ID NO: 542 of CAR123-2;

(iii) a LC CDR1 of SEQ ID NO: 533, LC CDR2 of SEQ ID NO: 538 and LC CDR3of SEQ ID NO: 543 of CAR123-3;

(iv) a LC CDR1 of SEQ ID NO: 534, LC CDR2 of SEQ ID NO: 539 and LC CDR3of SEQ ID NO: 544 of CAR123-4; and/or

-   (2) one, two or three heavy chain (HC) CDRs chosen from one of the    following:

(i) a HC CDR1 of SEQ ID NO:516, HC CDR2 of SEQ ID NO: 521 and HC CDR3 ofSEQ ID NO: 526 of CAR123-1;

(ii) a HC CDR1 of SEQ ID NO: 517, HC CDR2 of SEQ ID NO: 522 and HC CDR3of SEQ ID NO: 527 of CAR123-2;

(iii) a HC CDR1 of SEQ ID NO: 518, HC CDR2 of SEQ ID NO: 523 and HC CDR3of SEQ ID NO: 528 of CAR123-3;

(iv) a HC CDR1 of SEQ ID NO: 519, HC CDR2 of SEQ ID NO: 524 and HC CDR3of SEQ ID NO: 529 of CAR123-4.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.Headings, sub-headings or numbered or lettered elements, e.g., (a), (b),(i) etc, are presented merely for ease of reading. The use of headingsor numbered or lettered elements in this document does not require thesteps or elements be performed in alphabetical order or that the stepsor elements are necessarily discrete from one another. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 shows a graphical representation of CAR expression in JNL cellstransduced with anti-CD123 CAR constructs as evaluated by FACS andreported as the percent of cells showing signal above the level ofsignal in untransduced (CAR negative) cells using Protein L as adetection reagent.

FIGS. 2A, 2B, and 2C show graphical representations of CD123 CARactivity in JNL cells. Anti-CD123 CAR constructs were evaluated foractivity using a Jurkat cell line containing the luciferase reporterdriven by the NFAT promoter (termed JNL cells). CAR activity is measuredas activation of this NFAT-driven reporter.

FIG. 3 shows a graphical representation of CD123 CAR expression inprimary T-cells. Percentage of cells transduced (expressing theanti-CD123 CAR on the cell surface) and their relative fluorescenceintensity of expression were determined by flow cytometric analysis on aBD LSRFortessa or BD-FACSCanto using Protein L as a detection reagent.Gating histogram plots of relative fluorescent intensity from that FACSfor signal above unstained cells shows the percentage of transduced Tcells. Transduction resulted in a range of CAR positive cells from12-42%.

FIG. 4 shows a graphical representation of CD123-CART-mediated cellkilling. T cell killing was directed towards CD123-expressing MOLM13acute myelogenous leukemia cells stably expressing luciferase.Untransduced T cells were used to determine non-specific backgroundkilling levels. The cytolytic activities of CART-CD123 were measuredover a range of effector:target cell ratios of 4:1 and 2-fold downwarddilutions of T cells where effectors were defined as T cells expressingthe anti-CD123 chimeric receptor. Assays were initiated by mixing anappropriate number of T cells with a constant number of targets cells.After 20 hours luciferase signal was measured using the Bright-Glo™Luciferase Assay on the EnVision instrument.

FIGS. 5A and 5B show transduction efficiency of T cells with CD123-CARs.FIG. 5A shows transduction efficiency of T cells with 1172 and 1176.FIG. 5B shows transduction efficiency of T cells with CD123 CARs 2-4.

FIG. 6 shows flow cytometry of CD123 CARs 2-4 and 1172 and 1176 todetermine the CD4:CD8 ratio.

FIG. 7 (provided in FIGS. 7-1, 7-2, and 7-3) shows the degranulation ofCD123 CARs 2-4 and 1172 and 1176 upon exposure to CD123+ tumor cells.

FIG. 8 shows a graphical representation of a luciferase assay to assesscytotoxicity of CART cells (NVS 2-4, 1172 and 1176 clones) towards tumortarget cells (MOLM14).

FIG. 9 shows a comparison of tumor burden in NSG mice injected withluciferase expressing MOLM14 cells at D6 (before CART injection) and atday 13 (6 days post injection with NVS 2-4, 1172 or 1176 clones) or atday 20.

FIG. 10 shows expression of CD30 and CD123 in the Hodgkin's Lymphomacell line HDLM-2.

FIGS. 11A and 11B show CD123 CAR expression in T-cells transduced withpELNS anti-CD123-41BB-CD3 via detection of dsRed, a surrogate marker ofCD123 by T2A coexpression. dsRed was detected via flow cytometry.

FIG. 12 shows Q-PCR on CD123 expression in 4 Hodgkin's Lymphoma celllines (HDLM2, KMH2, L428, SUPHD1), CD123+ MOLM14 (positive control) andA375 (negative control). GUSB was used as a housekeeping gene. The Ctthreshold was 40.

FIG. 13 shows the effect of IL3 antibody on an in vivo model forHodgkin's Lymphoma. NOD-SCID-γ-chain KO mice that overexpress humancytokines including IL-3 (NSG-S mice) were engrafted with theluciferase-expressing HDLM-2 cell line. After i.v. injection, theneoplastic cells progressively formed disseminated soft tissue masses.Serial injections of a neutralizing anti-IL3 antibody slowed the growthof tumor.

FIG. 14 shows a CD107a degranulation assay. Untransducted T cells (UTD)or CART123 were incubated with HDLM-2 (CD123+ HL cell line) for 4 hoursat a ratio of 5 target cells to 1 T cells in the presence of anti-CD28,anti-CD49d antibodies and monensin. Anti-CD30 CAR T cells were used asadditional controls. CART123 but not UTD showed increase CD107adegranulation as detected by flow cytometry.

FIG. 15 shows a graphical representation of the degranulation assayshown in FIG. 20.

FIG. 16 demonstrates that CD123 CAR expressing T cells show asignificant increase in intra-cytoplasmatic cytokine production (IL-2,TNF).

FIG. 17 demonstrates CART123, but not UTD, showed a dose dependentkilling as shown by decrease in bioluminesce emission in a luciferasebased 24 hr killing assay. T cells were co-incubated with luciferase+HDML-2 cells at different rations (0.3:1-10:1).

FIG. 18 shows CART123 and CART30 cells but not UTD showed robustproliferation when co-cultured with Hodgkin Lymphoma cell linesaproliferation assay. T cells were incubated with HL cell lines (CD123+)or controls (Jurkat CD123−, MOLM-14 CD123+) or PMA/Ionomycin (positivecontrol) or cell media (negative control) in a 5-day T cellproliferation assay.

FIG. 19, comprising FIGS. 19A, 19B, 19C, and 19D show CART123 inducingrobust production of IFNg (FIG. 19A), IL-2 (FIG. 19B), TNFa (FIG. 19C),and MIP1b (FIG. 19D) luminex MFI are shown.

FIG. 20 shows a schematic representation of an in vivo model of HodgkinLymphoma. 1 million luciferase+HDLM-2 cells were injected i.v. on day 0.Serial bioluminescent imaging (BLI) demonstrated was then performed toobserve tumor level. A low level of tumor was observed on day 7, whichwas followed by gradual increase in tumor burden over approximately 6weeks, reproducing the indolent nature of the human disease. At day 43when the tumor burden was 20-fold higher than baseline, mice weretreated with 1.5 million CART123 cells or control T cells.

FIG. 21 shows BLI to detect HDLM-2 at days post-injection in micetreated with CART123 cells, control T cells, or untreated, according tothe schematic in FIG. 28.

FIG. 22 shows survival of mice treated according to the schematic shownin FIG. 28, CART123 induced complete and durable eradication ofdisseminated tumor within 14 days, leading to 100% relapse-free and 100%overall survival at 6 months.

FIG. 23 shows an association between tumor elimination and extensive CART cell expansion as detected by flow cytometry in serial peripheralblood bleedings. The expanded T cells were approximately 50% CD8 and 50%CD4 cells. The T cell number contracted over time as tumor burdendecreased.

FIGS. 24A, 24B, and 24C are bar graphs showing the cytokine productionfrom T cells expressing CAR123 constructs (CD123-2, CD123-3, andCD123-4) when cultured with target cells MOLM13, PL21, and U87 at theindicated ratios. FIG. 24A shows production of IL-2; FIG. 24B shows theproduction of IFN-gamma; and FIG. 24C shows the production of TNF-alpha.

FIG. 25 is a bar graph showing the proliferation capacity oflentivirally transduced T cells expressing CAR123-2 (LV CAR123-2) orcontrol anti-GH when cultured in the presence of target cells MOLM13(which express CD123), K562-human CD123 (which express human CD123), orK562 (which do not express CD123).

FIGS. 26A, 26B, and 26C show the ability of lentivirally transduced Tcells expressing CAR123 CAR (LV CAR123-2) or control (Anti-GH), oruntransduced cells (UTD) to kill target cells at various targetcell:effector cell ratios, where the target cells are CD123-expressingMOLM13 (FIG. 26A) or PL21 (FIG. 26B) cells, or CD123-negative U87 cells(FIG. 26C).

FIGS. 27A and 27B are graphs showing cytokine production fromlentivirally transduced T cells expressing either a CAR123 (LV CAR123-2)or control (Anti-GH) when cultured in the present of target cells MOLM13(which express CD123) and U87 (which do not express CD123). FIG. 27Ashows the cytokine production of IFNγ and TNFα; FIG. 27B shows thecytokine production of IL-2.

FIG. 28 shows the tumor burden in an in vivo mouse model of Hodgkinlymphoma, where the mice bearing tumors were administered T cellsexpressing various CAR123 constructs: 1172, 1176, NVS2 (also referred toherein as CAR123-2), NVS3 (also referred to herein as CAR123-3), andNVS4 (also referred to herein as CAR123-4). Tumor burden at day 6, day14, day 20, and day 27 were measured and represented by bioluminescentimaging (BLI).

FIG. 29 shows the tumor burden in an in vivo mouse model, where the micebearing tumors were administered T cells expressing CAR123-2 introducedfrom a lentiviral vector (CART123 LV), CAR123-2 introduced as RNA(CART123 RNA (NVS)), and a tool CAR123, introduced as RNA (CART123 RNA(Penn)). Untransduced T cells were used as control (UTD). Tumor burdenis represented by bioluminescent imaging (BLI) units.

FIGS. 30A, 30B, 30C, and 30D show bioluminescent imaging of the tumorsin an in vivo mouse model. FIG. 30A shows the mice administereduntransduced cells; FIG. 30B shows the mice administered the T cellsexpressing RNA CAR123-2; FIG. 30C shows the mice administeredlentivirally transduced T cells expressing CAR123-2; FIG. 30D shows themice administered T cells expressing RNA tool CAR123.

FIGS. 31A, 31B, 31C, 31D, 31E, and 31F show CD123 is highly expressed inCD19-neg B-cell acute lymphoblastic leukemia relapses occurring afterCART19 treatment. FIG. 31A shows expression of CD123 compared to CD19 in42 relapsing/refractory ALL samples. FIG. 31B shows CD123 and CD19co-expression in B-ALL blasts. Gated on blasts (SSC low, singlet, live,CD45dim). FIG. 31C shows the gating strategy for the leukemia stem cell(LSC). CD123 is highly expressed in this subset. FIG. 31D shows CD123and CD19 co-expression and results from FISH analysis. FIGS. 31E and 31Fshow the comparison of CD19 and CD123 expression at baseline or afterrelapse.

FIGS. 32A, 32B, 32C, 32D, 32E, and 32F show results from various invitro assays using T cells expressing a CD19 CAR (CAR19) or a CD123 CAR(CAR123). FIG. 32A shows CD19 and CD123 expression; FIG. 32B shows aCD107a degranulation assay; FIG. 32C shows the capability for targetedcell killing; FIGS. 32D and E shows proliferation capacity; FIG. 32Fshows cytokine production for the indicated cytokines.

FIGS. 33A, 33B, and 33C show that CART cells expressing CD19 CAR (CAR19)or CD123 CAR (CAR123) had an anti-tumor effect in an in vivo mousemodel. FIG. 33A shows the tumor burden represented by bioluminescentimaging; FIG. 33B shows the overall survival curve of mice receivingCART therapy; and FIG. 33C shows the expansion of CART123 cells in theperipheral blood.

FIGS. 34A, 34B, 34C, 34D, 34E, and 34F show that CART123 is active in anin vivo mouse model of antigen-loss relapse. FIG. 34A shows theexperimental schema; FIG. 34B shows disease progression as representedby bioluminescent imaging in baseline and relapse disease with respectto CD19 expression (top graph) and in response to treatment with CART19therapy (bottom graph); FIG. 34C shows bioluminescent images of miceadministered untransduced T cells or CART19 cells; FIG. 34D shows theexperimental schema for treating with CART19 or CART123; FIG. 34E showsthe disease progression; and FIG. 34F shows the overall survival of thetreated mice.

FIGS. 35A, 35B, and 35C show ALL-CART interactions in skull bone marrowof xenograft mice. FIG. 35A shows the experimental schema; FIG. 35Bshows representative multiphoton XY plane images of CART19 cells andCART123 cells interacting with ALL tumor engineered to express eitherCD19 and CD123 or CD123 alone (motile cells are indicated in dashedcircles, non-motile cells are indicated with the arrows); and FIG. 35Cis a graphic representation of the microscopy images.

FIGS. 36A, 36B, and 36C show the prevention of CD19-neg relapses usingCART19 and CART123. FIG. 36A shows the experimental schema; FIG. 36Bshows the disease progression (tumor burden as represented by BLI) ofmice treated with untransduced T cells (top graph), CART19 (middlegraph), or the combination of CART19 and CART123 (bottom graph); andFIG. 36C shows the overall survival from this experiment.

FIGS. 37A and 37B show T cells expressing both CAR19 and CAR123 (FIG.37A) and the results from a degranulation assay (FIG. 37B).

FIGS. 38A and 38B show characterization of ALL blasts. FIG. 38A showsexpression of various markers CD19, CD123, CD10, CD34, and CD20; andFIG. 38B shows the gating strategy for sorting CD19−CD123+ cells.

FIGS. 39A, 39B, 39C, and 39D show anti-leukemia activity of CART123.FIG. 39A shows the expression of CD19 and CD123 on the NALM6 cells; FIG.39B shows the tumor burden (as represented by BLI) in response to CART19or CART123 therapy; FIG. 39C shows the overall survival of miceadministered CART19 or CART123; and FIG. 39D shows the overall survivalof mice administered varying doses of CART123.

FIGS. 40A and 40B show the characterization of the in vivo model ofantigen-loss relapse. FIG. 40A shows the expression of CD123 in CD19negative relapse disease; and FIG. 40B shows the degranulation assay ofCART19 or CART123 cells when cultured with baseline or relapse cells invitro.

FIGS. 41A, 41B and 41C show the expression of PD1 L1 or on primary AMLcells (FIG. 41A), and PD1 (FIG. 41B) and TIM3 (FIG. 41C) on T cells.

FIGS. 42A, 42B, 42C, and 42D show the expression of immune checkpointsPD1 (FIG. 42A), TIM3 (FIG. 42B), LAG3 (FIG. 42C), and CTLA4 (FIG. 42D)on CD8+ T cells.

FIG. 43 shows flow cytometry analysis of TIM3 expression on AML baseline(left) and relapsed (right) samples.

FIG. 44 shows the tumor burden (represented by bioluminescent imaging,photons/sec) after CART123 treatment compared to treatment withuntransduced (UTD) cells.

FIG. 45 shows the expression of TIM3 and PD1 on T cells in xenograftsafter CD123 treatment in remission or after relapse.

FIGS. 46A and 46B show the characterization of bone marrow from relapsedmice after coculture with checkpoint inhibitors, e.g., inhibitors ofPD1, TIM3, a combination of PD1 and TIM3 inhibitors, or a combination ofTIM3 and CEACAM-1 inhibitors. The percentage of T cells expressingGMCSF, IFNg, Ki67, and MIP1b was detected in the absence (FIG. 46A) orin the presence of PMA/IONO (FIG. 46B).

FIG. 47 shows the experimental schema for treating AML xenografts withuntransduced cells and PD1 inhibitors or TIM3 inhibitors.

FIG. 48 shows the tumor burden (represented by BLI, photons/sec) for theexperiment depicted in FIG. 47.

FIG. 49 shows the experimental schema for treating AML xenografts withCART123 in combination with TIM3 and/or PD1 inhibitors.

FIGS. 50A, 50B, 50C, and 50D show the tumor burden (represented by BLI,photons/sec) for the experiment depicted in FIG. 49. FIG. 50A comparesCART123 with the combination of CART123 and PD1 inhibitor; FIG. 50Bcompares CART123 with the combination of CART123 and PD1 inhibitor andTIM3 inhibitor; FIG. 50C compares CART123 with the combination ofCART123 and TIM3 inhibitor, and FIG. 50D shows a representative plot forthe data presented in FIGS. 50A-C.

FIGS. 51A, 51B, 51C, 51D, and 51E show the various configurations on asingle vector, e.g., where the U6 regulated shRNA is upstream ordownstream of the EF1 alpha regulated CAR encoding elements. In theexemplary constructs depicted in FIGS. 51A and 51B, the transcriptionoccurs through the U6 and EF1 alpha promoters in the same direction. Inthe exemplary constructs depicted in FIGS. 51C and 51D, thetranscription occurs through the U6 and EF1 alpha promoters in differentdirections. In FIG. 51E, the shRNA (and corresponding U6 promoter) is ona first vector, and the CAR (and corresponding EF1 alpha promoter) is ona second vector.

FIG. 52 depicts the structures of two exemplary RCAR configurations. Theantigen binding members comprise an antigen binding domain, atransmembrane domain, and a switch domain. The intracellular bindingmembers comprise a switch domain, a co-stimulatory signaling domain anda primary signaling domain. The two configurations demonstrate that thefirst and second switch domains described herein can be in differentorientations with respect to the antigen binding member and theintracellular binding member. Other RCAR configurations are furtherdescribed herein.

FIG. 53 shows that the proliferation of CAR-expressing, transduced Tcells is enhanced by low doses of RAD001 in a cell culture system. CARTswere co-cultured with Nalm-6 cells in the presence of differentconcentrations of RAD001. The number of CAR-positive CD3-positive Tcells (black) and total T cells (gray) was assessed after 4 days ofco-culture.

FIG. 54 depicts tumor growth measurements of NALM6-luc cells with dailyRAD001 dosing at 0.3, 1, 3, and 10 mg/kg (mpk) or vehicle dosing.Circles denote the vehicle; squares denote the 10 mg/kg dose of RAD001;triangles denote the 3 mg/kg dose of RAD001, inverted triangles denotethe 1 mg/kg dose of RAD001; and diamonds denote the 0.3 mg/kg dose ofRAD001.

FIGS. 55A and 55B show pharmacokinetic curves showing the amount ofRAD001 in the blood of NSG mice with NALM6 tumors. FIG. 55A shows day 0PK following the first dose of RAD001. FIG. 55B shows Day 14 PKfollowing the final RAD001 dose. Diamonds denote the 10 mg/kg dose ofRAD001; squares denote the 1 mg/kg dose of RAD001; triangles denote the3 mg/kg dose of RAD001; and x's denote the 10 mg/kg dose of RAD001.

FIGS. 56A and 56B show in vivo proliferation of humanized CD19 CARTcells with and without RAD001 dosing. Low doses of RAD001 (0.003 mg/kg)daily lead to an enhancement in CAR T cell proliferation, above thenormal level of huCAR19 proliferation. FIG. 56A shows CD4⁺ CAR T cells;FIG. 56B shows CD8⁺ CAR T cells. Circles denote PBS; squares denotehuCTL019; triangles denote huCTL019 with 3 mg/kg RAD001; invertedtriangles denote huCTL019 with 0.3 mg/kg RAD001; diamonds denotehuCTL019 with 0.03 mg/kg RAD001; and circles denote huCTL019 with 0.003mg/kg RAD001.

FIG. 57 is a vector map of a lentiviral vector for expressing a CAR123and CD20 in a strategy for terminating CART123 activity, where theCAR123 sequence and the CD20 sequence are linked by a P2A sequence.

FIG. 58 shows the results of a CD107 degranulation assay comparingCART123 to CART123 P2A CD20 cells.

FIGS. 59A, 59B, 59C, and 59D show the production of GM-CSF (FIG. 59A),TNFalpha (FIG. 59B), IFNgamma (FIG. 59C), and IL-2 (FIG. 59D) by CART123cells compared to CART123 P2A CD20 cells.

FIG. 60 shows the cytotoxic capability (as represented by % specificlysis) of CART123 cells compared to CART123 P2A CD20 cells whenincubated at the indicated effector:target cell ratios.

FIGS. 61A and 61B show T cell depletion after treatment with theindicated doses of rituximab on CART123 cells (FIG. 61A) and CART123 P2ACD20 cells (FIG. 61B).

FIG. 62 shows the T cell depletion after treatment with the indicateddoses of rituximab on CART123 P2A CD20 cells in the presence of 15%complement.

FIGS. 63A and 63B demonstrate the strategy for a dual CART cellexpressing both a CAR19 and a CAR123. FIG. 63A is a vector mapcontaining a CAR19 and a CAR123, linked through a P2A sequence. FIG. 63Bshows the percentage of cells expressing CAR19, CAR123 (Q3), and bothCAR19 (Q1) and CAR123 (Q2).

FIGS. 64A and 64B demonstrate the strategy for a split CART cell thatexpresses both a CAR19 and a CAR123. FIG. 64A is a vector map containinga CAR19 and a CAR123, linked through a P2A sequence. The CAR19 includesa CD3zeta domain while the CAR123 includes a 4-1BB domain. FIG. 64Bshows the percentage of cells expressing CAR19, CAR123 (Q3), and bothCAR19 (Q1) and CAR123 (Q2).

FIG. 65 shows the expression of CD123 on primary HSC, AML, and BPDCNsamples.

FIGS. 66A and 66B show a cytotoxicity assay for CART123 clones 32716(FIG. 66A) and 26292 (FIG. 66A) when incubated in the presence ofCD123-expressing target cells at the indicated effector:target ratios.

FIG. 67 shows a CD107 degranulation assay for CART123 cells whenincubated with HSCs compared to BPDCN.

FIGS. 68A and 68B show CD123 expression on mononuclear cells from theblood of a patient with mast cell leukemia/systemic mastocytosis asdetected by staining by isotype antibody (FIG. 68A) as control or CD123antibody (FIG. 68B).

FIGS. 69A and 69B show a CD107 degranulation assay where CART123 cellswere cultured alone with PMA and ionomycin (FIG. 69A) or withmononuclear cells from the blood of a patient with mast cellleukemia/systemic mastocytosis (FIG. 69B).

FIG. 70 shows the experimental schema for subject cohort 1 in a clinicalstudy for administering RNA CART123 in subjects with refractory orrelapsed AML.

FIG. 71 shows the experimental schema for subject cohort 2 in a clinicalstudy for administering RNA CART123 in subjects with refractory orrelapsed AML. These patients receive doses of a lymphodepletingchemotherapy in addition to the CART therapy.

FIGS. 72A, 72B, and 72C show the results of T cell ablation usinganti-CD52 antibody alemtuzumab after treatment with lentivirallytransduced CART123 cells in a MOLM14 xenograft model. CART123 cells oruntransduced (UTD) cells were administered to mice at week 1.Alemtuzumab was administered at week 2, week 3, or week 4 to mice thatreceived CART123 cells, and tumor burden was assessed for 24 weeks. A“MOLM14” rechallenge was introduced at 12 weeks. FIG. 72A shows tumorburden as detected by biolumionescent imaging (photons/sec); FIG. 72Bshows the percent of CART cells detected in peripheral blood; and FIG.72C shows the overall survival.

FIGS. 73A and 73B show the results of T cell ablation using alemtuzumabafter treatment with lentivirally transduced CART123 cells in apediatric AML xenograft model. CART123 cells or untransduced (UTD) cellswere administered to mice 6 weeks after primary AML cell engraftment(week 0). Alemtuzumab was administered at week 4 to mice that receivedCART123 treatment. FIG. 73A shows tumor burden as detected bybiolumionescent imaging (photons/sec); FIG. 73B shows the percent ofCART cells detected in peripheral blood.

FIGS. 74A and 74B show the analysis of AML cells in the spleen (FIG.74A) and the bone marrow (FIG. 74B) after CART123 and alemtuzumabtreatment, as performed in FIGS. 73A-73B.

FIG. 75 shows the comparison of the three termination strategies: (1)CART cell ablation by alemtuzumab treatment; (2) CAR/CD20 co-expressionin T cells and ablation by rituximab; and (3) RNA-electroporated CARTcells, on tumor burden (represented by bioluminescent imaging;photons/sec) in the AML xenograft model.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa recombinant polypeptide construct comprising at least an extracellularantigen binding domain, a transmembrane domain and a cytoplasmicsignaling domain (also referred to herein as “an intracellular signalingdomain”) comprising a functional signaling domain derived from astimulatory molecule as defined below. In some embodiments, the domainsin the CAR polypeptide construct are in the same polypeptide chain,e.g., comprise a chimeric fusion protein. In some embodiments, thedomains in the CAR polypeptide construct are not contiguous with eachother, e.g., are in different polypeptide chains, e.g., as provided inan RCAR as described herein.

In one aspect, the stimulatory molecule of the CAR is the zeta chainassociated with the T cell receptor complex. In one aspect, thecytoplasmic signaling domain comprises a primary signaling domain (e.g.,a primary signaling domain of CD3-zeta). In one aspect, the cytoplasmicsignaling domain further comprises one or more functional signalingdomains derived from at least one costimulatory molecule as definedbelow. In one aspect, the costimulatory molecule is chosen from 4-1BB(i.e., CD137), CD27, ICOS, and/or CD28. In one aspect, the CAR comprisesa chimeric fusion protein comprising an extracellular antigenrecognition domain, a transmembrane domain and an intracellularsignaling domain comprising a functional signaling domain derived from astimulatory molecule. In one aspect, the CAR comprises a chimeric fusionprotein comprising an extracellular antigen recognition domain, atransmembrane domain and an intracellular signaling domain comprising afunctional signaling domain derived from a co-stimulatory molecule and afunctional signaling domain derived from a stimulatory molecule. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising two functional signalingdomains derived from one or more co-stimulatory molecule(s) and afunctional signaling domain derived from a stimulatory molecule. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more co-stimulatory molecule(s)and a functional signaling domain derived from a stimulatory molecule.In one aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen recognition domain, wherein the leader sequence isoptionally cleaved from the antigen recognition domain (e.g., aa scFv)during cellular processing and localization of the CAR to the cellularmembrane.

A CAR that comprises an antigen binding domain (e.g., a scFv, a singledomain antibody, or TCR (e.g., a TCR alpha binding domain or TCR betabinding domain)) that specifically binds a specific tumor marker X,wherein X can be a tumor marker as described herein, is also referred toas XCAR. For example, a CAR that comprises an antigen binding domainthat specifically binds CD123 is referred to as CD123 CAR. The CAR canbe expressed in any cell, e.g., an immune effector cell as describedherein (e.g., a T cell or an NK cell).

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

As used herein, the terms “alpha subunit of the IL-3 receptor,” “IL3Rα,”“CD123,” “IL3Rα chain” and “IL3Rα subunit” refer interchangeably to anantigenic determinant known to be detectable on leukemia precursorcells. The human and murine amino acid and nucleic acid sequences can befound in a public database, such as GenBank, UniProt and Swiss-Prot. Forexample, the amino acid sequence of human IL3Rα can be found atAccession No. NP 002174 and the nucleotide sequence encoding of thehuman IL3Rα can be found at Accession No. NM 005191. In one aspect theantigen-binding portion of the CAR recognizes and binds an epitopewithin the extracellular domain of the CD123 protein. In one aspect, theCD123 protein is expressed on a cancer cell. As used herein, “CD123”includes proteins comprising mutations, e.g., point mutations,fragments, insertions, deletions and splice variants of full lengthwild-type CD123.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of an intactantibody, or recombinant variants thereof, and refers to the antigenbinding domain, e.g., an antigenic determining variable region of anintact antibody, that is sufficient to confer recognition and specificbinding of the antibody fragment to a target, such as an antigen.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)₂, and Fv fragments, scFv antibody fragments, linearantibodies, single domain antibodies such as sdAb (either VL or VH),camelid VHH domains, and multi-specific antibodies formed from antibodyfragments such as a bivalent fragment comprising two Fab fragmentslinked by a disulfide brudge at the hinge region, and an isolated CDR orother epitope binding fragments of an antibody. An antigen bindingfragment can also be incorporated into single domain antibodies,maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, NatureBiotechnology 23:1126-1136, 2005). Antigen binding fragments can also begrafted into scaffolds based on polypeptides such as a fibronectin typeIII (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectinpolypeptide minibodies).

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked via a short flexible polypeptide linker, and capableof being expressed as a single chain polypeptide, and wherein the scFvretains the specificity of the intact antibody from which it is derived.Unless specified, as used herein an scFv may have the VL and VH variableregions in either order, e.g., with respect to the N-terminal andC-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL.

The term “complementarity determining region” or “CDR,” as used herein,refers to the sequences of amino acids within antibody variable regionswhich confer antigen specificity and binding affinity. For example, ingeneral, there are three CDRs in each heavy chain variable region (e.g.,HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variableregion (LCDR1, LCDR2, and LCDR3). The precise amino acid sequenceboundaries of a given CDR can be determined using any of a number ofwell-known schemes, including those described by Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (“Kabat” numberingscheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numberingscheme), or a combination thereof. Under the Kabat numbering scheme, insome embodiments, the CDR amino acid residues in the heavy chainvariable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3); and the CDR amino acid residues in the light chainvariable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments,the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2),and 95-102 (HCDR3); and the CDR amino acid residues in the VL arenumbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combinedKabat and Chothia numbering scheme, in some embodiments, the CDRscorrespond to the amino acid residues that are part of a Kabat CDR, aChothia CDR, or both. For instance, in some embodiments, the CDRscorrespond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; andamino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in aVL, e.g., a mammalian VL, e.g., a human VL.

The portion of the CAR composition of the invention comprising anantibody or antibody fragment thereof may exist in a variety of formswhere the antigen binding domain is expressed as part of a contiguouspolypeptide chain including, for example, a single domain antibodyfragment (sdAb), a single chain antibody (scFv) and a humanized or humanantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

As used herein, the term “binding domain” or “antibody molecule” (alsoreferred to herein as “anti-target (e.g., CD123) binding domain”) refersto a protein, e.g., an immunoglobulin chain or fragment thereof,comprising at least one immunoglobulin variable domain sequence. Theterm “binding domain” or “antibody molecule” encompasses antibodies andantibody fragments. In an embodiment, an antibody molecule is amultispecific antibody molecule, e.g., it comprises a plurality ofimmunoglobulin variable domain sequences, wherein a first immunoglobulinvariable domain sequence of the plurality has binding specificity for afirst epitope and a second immunoglobulin variable domain sequence ofthe plurality has binding specificity for a second epitope. In anembodiment, a multispecific antibody molecule is a bispecific antibodymolecule. A bispecific antibody has specificity for no more than twoantigens. A bispecific antibody molecule is characterized by a firstimmunoglobulin variable domain sequence which has binding specificityfor a first epitope and a second immunoglobulin variable domain sequencethat has binding specificity for a second epitope.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (κ) and lambda (λ) light chains refer tothe two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample, or might be macromolecule besides a polypeptide. Sucha biological sample can include, but is not limited to a tissue sample,a tumor sample, a cell or a fluid with other biological components.

The term “anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in the number of metastases, an increase in life expectancy,decrease in tumor cell proliferation, decrease in tumor cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies ofthe invention in prevention of the occurrence of tumor in the firstplace.

The term “anti-cancer effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of cancer cells, adecrease in the number of metastases, an increase in life expectancy,decrease in cancer cell proliferation, decrease in cancer cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-cancer effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies inprevention of the occurrence of cancer in the first place.

The term “anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in tumor cell proliferation, or a decrease in tumor cellsurvival.

The term “autologous” refers to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically.

The term “xenogeneic” refers to a graft derived from an animal of adifferent species. The term “apheresis” as used herein refers to theart-recognized extracorporeal process by which the blood of a donor orpatient is removed from the donor or patient and passed through anapparatus that separates out selected particular constituent(s) andreturns the remainder to the circulation of the donor or patient, e.g.,by retransfusion. Thus, in the context of “an apheresis sample” refersto a sample obtained using apheresis.

The term “combination” refers to either a fixed combination in onedosage unit form, or a combined administration where a compound of thepresent invention and a combination partner (e.g. another drug asexplained below, also referred to as “therapeutic agent” or “co-agent”)may be administered independently at the same time or separately withintime intervals, especially where these time intervals allow that thecombination partners show a cooperative, e.g. synergistic effect. Thesingle components may be packaged in a kit or separately. One or both ofthe components (e.g., powders or liquids) may be reconstituted ordiluted to a desired dose prior to administration. The terms“co-administration” or “combined administration” or the like as utilizedherein are meant to encompass administration of the selected combinationpartner to a single subject in need thereof (e.g. a patient), and areintended to include treatment regimens in which the agents are notnecessarily administered by the same route of administration or at thesame time. The term “pharmaceutical combination” as used herein means aproduct that results from the mixing or combining of more than oneactive ingredient and includes both fixed and non-fixed combinations ofthe active ingredients. The term “fixed combination” means that theactive ingredients, e.g. a compound of the present invention and acombination partner, are both administered to a patient simultaneouslyin the form of a single entity or dosage. The term “non-fixedcombination” means that the active ingredients, e.g. a compound of thepresent invention and a combination partner, are both administered to apatient as separate entities either simultaneously, concurrently orsequentially with no specific time limits, wherein such administrationprovides therapeutically effective levels of the two compounds in thebody of the patient. The latter also applies to cocktail therapy, e.g.the administration of three or more active ingredients.

The term “cancer” refers to a disease characterized by the rapid anduncontrolled growth of aberrant cells. Cancer cells can spread locallyor through the bloodstream and lymphatic system to other parts of thebody. Examples of various cancers are described herein and include butare not limited to, breast cancer, prostate cancer, ovarian cancer,cervical cancer, skin cancer, pancreatic cancer, colorectal cancer,renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lungcancer and the like. The terms “tumor” and “cancer” are usedinterchangeably herein, e.g., both terms encompass solid and liquid,e.g., diffuse or circulating, tumors. As used herein, the term “cancer”or “tumor” includes premalignant, as well as malignant cancers andtumors.

“Derived from” as that term is used herein, indicates a relationshipbetween a first and a second molecule. It generally refers to structuralsimilarity between the first molecule and a second molecule and does notconnotate or include a process or source limitation on a first moleculethat is derived from a second molecule. For example, in the case of anintracellular signaling domain that is derived from a CD3zeta molecule,the intracellular signaling domain retains sufficient CD3zeta structuresuch that is has the required function, namely, the ability to generatea signal under the appropriate conditions. It does not connotate orinclude a limitation to a particular process of producing theintracellular signaling domain, e.g., it does not mean that, to providethe intracellular signaling domain, one must start with a CD3zetasequence and delete unwanted sequence, or impose mutations, to arrive atthe intracellular signaling domain.

The phrase “disease associated with expression of CD123” as used hereinincludes but is not limited to, a disease associated with expression ofCD123 or condition associated with a cell which expresses CD123 (e.g.,wild-type or mutant CD123) including, e.g., a proliferative disease suchas a cancer or malignancy; a precancerous condition such as amyelodysplasia, a myelodysplastic syndrome or a preleukemia; or anon-cancer related indication associated with a cell which expressesCD123 (e.g., wild-type or mutant CD123). In one aspect, a cancerassociated with expression of CD123 (e.g., wild-type or mutant CD123) isa hematological cancer. In one aspect, the disease includes AML, ALL,hairy cell leukemia, Prolymphocytic leukemia, Chronic myeloid leukemia(CML), Hodgkin lymphoma, Blastic plasmacytoid dendritic cell neoplasm,lymphoblastic B-cell leukemia (B-cell acute lymphoid leukemia, BALL),acute lymphoblastic T-cell leukemia (T-cell acute lymphoid leukemia(TALL); myelodysplastic syndrome; a myeloproliferative neoplasm; ahistiocytic disorder (e.g., a mast cell disorder or a blasticplasmacytoid dendritic cell neoplasm); a mast cell disorder, e.g.,systemic mastocytosis or mast cell leukemia, and the like. Furtherdisease associated with expression of CD123 expression include, but arenot limited to, e.g., atypical and/or non-classical cancers,malignancies, precancerous conditions or proliferative diseasesassociated with expression of CD123. Non-cancer related indicationsassociated with expression of CD123 may also be included.

In some embodiments, the tumor antigen (e.g., CD123− orCD19−)-expressing cell expresses, or at any time expressed, mRNAencoding the tumor antigen. In an embodiment, the tumor antigen (e.g.,CD123− or CD19−)-expressing cell produces the tumor antigen protein(e.g., wild-type or mutant), and the tumor antigen protein may bepresent at normal levels or reduced levels. In an embodiment, the tumorantigen (e.g., CD123− or CD19−)-expressing cell produced detectablelevels of a tumor antigen protein at one point, and subsequentlyproduced substantially no detectable tumor antigen protein.

The term “conservative sequence modifications” refers to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody or antibody fragment containing theamino acid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody or antibody fragment of the invention by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative substitutions are ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within a CAR of the invention can bereplaced with other amino acid residues from the same side chain familyand the altered CAR can be tested using the functional assays describedherein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognateligand thereby mediating a signal transduction event, such as, but notlimited to, signal transduction via the TCR/CD3 complex. Stimulation canmediate altered expression of certain molecules, such as downregulationof TGF-β, and/or reorganization of cytoskeletal structures, and thelike.

The term “stimulatory molecule,” refers to a molecule expressed by a Tcell that provides the primary cytoplasmic signaling sequence(s) thatregulate primary activation of the TCR complex in a stimulatory way forat least some aspect of the T cell signaling pathway. In one aspect, theprimary signal is initiated by, for instance, binding of a TCR/CD3complex with an MHC molecule loaded with peptide, and which leads tomediation of a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. A primarycytoplasmic signaling sequence (also referred to as a “primary signalingdomain”) that acts in a stimulatory manner may contain a signaling motifwhich is known as immunoreceptor tyrosine-based activation motif orITAM. Examples of an ITAM containing primary cytoplasmic signalingsequence that is of particular use in the invention includes, but is notlimited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma,CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as“ICOS”), FcεRI, CD66d, DAP10 and DAP12. In a specific CAR of theinvention, the intracellular signaling domain in any one or more CARS ofthe invention comprises an intracellular signaling sequence, e.g., aprimary signaling sequence of CD3-zeta. In a specific CAR of theinvention, the primary signaling sequence of CD3-zeta is the sequenceprovided as SEQ ID NO:9, or the equivalent residues from a non-humanspecies, e.g., mouse, rodent, monkey, ape and the like. In a specificCAR of the invention, the primary signaling sequence of CD3-zeta is thesequence as provided in SEQ ID NO:10, or the equivalent residues from anon-human species, e.g., mouse, rodent, monkey, ape and the like.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (MHC's) on its surface. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain can generate a signal that promotes an immune effector functionof the CAR containing cell, e.g., a CART cell or CAR-expressing NK cell.Examples of immune effector function, e.g., in a CART cell orCAR-expressing NK cell, include cytolytic activity and helper activity,including the secretion of cytokines. In embodiments, the intracellularsignal domain transduces the effector function signal and directs thecell to perform a specialized function. While the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CAR-expressing immuneeffector cell, e.g., CART cell or CAR-expressing NK cell, a primaryintracellular signaling domain can comprise a cytoplasmic sequence of aT cell receptor, and a costimulatory intracellular signaling domain cancomprise cytoplasmic sequence from co-receptor or costimulatorymolecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22,CD79a, CD79b, CD278 (“ICOS”), FcεRI, CD66d, DAP10, and DAP12.

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBan Acc. No. BAG36664.1, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain that are sufficient to functionally transmit aninitial signal necessary for T cell activation. In one aspect thecytoplasmic domain of zeta comprises residues 52 through 164 of GenBankAcc. No. BAG36664.1 or the equivalent residues from a non-human species,e.g., mouse, rodent, monkey, ape and the like, that are functionalorthologs thereof. In one aspect, the “zeta stimulatory domain” or a“CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO:9.In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatorydomain” is the sequence provided as SEQ ID NO:10.

The term “costimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient immune response. Costimulatory moleculesinclude, but are not limited to an a MHC class I molecule, TNF receptorproteins, Immunoglobulin-like proteins, cytokine receptors, integrins,signaling lymphocytic activation molecules (SLAM proteins), activatingNK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

A costimulatory intracellular signaling domain refers to theintracellular portion of a costimulatory molecule. The intracellularsignaling domain can comprise the entire intracellular portion, or theentire native intracellular signaling domain, of the molecule from whichit is derived, or a functional fragment thereof.

The term “4-1BB” refers to a member of the TNFR superfamily with anamino acid sequence provided as GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like; and a “4-1BB costimulatory domain” is definedas amino acid residues 214-255 of GenBank accno. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In one aspect, the “4-1BB costimulatorydomain” is the sequence provided as SEQ ID NO:7 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like.

“Immune effector cell,” as that term is used herein, refers to a cellthat is involved in an immune response, e.g., in the promotion of animmune effector response. Examples of immune effector cells include Tcells, e.g., alpha/beta T cells and gamma/delta T cells, B cells,natural killer (NK) cells, natural killer T (NKT) cells, mast cells, andmyeloic-derived phagocytes.

“Immune effector function or immune effector response,” as that term isused herein, refers to function or response, e.g., of an immune effectorcell, that enhances or promotes an immune attack of a target cell. E.g.,an immune effector function or response refers a property of a T or NKcell that promotes killing or the inhibition of growth or proliferation,of a target cell. In the case of a T cell, primary stimulation andco-stimulation are examples of immune effector function or response.

The term “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “vector” as used herein refers to any vehicle that can be usedto deliver and/or express a nucleic acid molecule. It can be a transfervector or an expression vector as described herein.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic, include but are not limited to, e.g., the LENTIVECTOR®gene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies and antibody fragments thereofare human immunoglobulins (recipient antibody or antibody fragment) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, a humanizedantibody/antibody fragment can comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. These modifications can further refine and optimize antibodyor antibody fragment performance. In general, the humanized antibody orantibody fragment thereof will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or a significant portion of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody or antibody fragment canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody orantibody fragment, where the whole molecule is of human origin orconsists of an amino acid sequence identical to a human form of theantibody or immunoglobulin.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid,” “polynucleotide,” or “nucleic acid molecule”refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or acombination of a DNA or RNA thereof, and polymers thereof in eithersingle- or double-stranded form. The term “nucleic acid” includes agene, cDNA or an mRNA. In one embodiment, the nucleic acid molecule issynthetic (e.g., chemically synthesized) or recombinant. Unlessspecifically limited, the term encompasses nucleic acids containinganalogues or derivatives of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, SNPs, and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The term “cancer associated antigen” or “tumor antigen” interchangeablyrefers to a molecule (typically a protein, carbohydrate or lipid) thatis expressed on the surface of a cancer cell, either entirely or as afragment (e.g., MHC/peptide), and which is useful for the preferentialtargeting of a pharmacological agent to the cancer cell. In someembodiments, a tumor antigen is a marker expressed by both normal cellsand cancer cells, e.g., a lineage marker, e.g., CD19 or CD123 on Bcells. In some embodiments, a tumor antigen is a cell surface moleculethat is overexpressed in a cancer cell in comparison to a normal cell,for instance, 1-fold over expression, 2-fold overexpression, 3-foldoverexpression or more in comparison to a normal cell. In someembodiments, a tumor antigen is a cell surface molecule that isinappropriately synthesized in the cancer cell, for instance, a moleculethat contains deletions, additions or mutations in comparison to themolecule expressed on a normal cell. In some embodiments, a tumorantigen will be expressed exclusively on the cell surface of a cancercell, entirely or as a fragment (e.g., MHC/peptide), and not synthesizedor expressed on the surface of a normal cell. In some embodiments, theCARs of the present invention includes CARs comprising an antigenbinding domain (e.g., antibody or antibody fragment) that binds to a MHCpresented peptide. Normally, peptides derived from endogenous proteinsfill the pockets of Major histocompatibility complex (MHC) class Imolecules, and are recognized by T cell receptors (TCRs) on CD8+ Tlymphocytes. The MHC class I complexes are constitutively expressed byall nucleated cells. In cancer, virus-specific and/or tumor-specificpeptide/MHC complexes represent a unique class of cell surface targetsfor immunotherapy. TCR-like antibodies targeting peptides derived fromviral or tumor antigens in the context of human leukocyte antigen(HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., JVirol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165;Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci TranslMed 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 201219(2):84-100). For example, TCR-like antibody can be identified fromscreening a library, such as a human scFv phage displayed library.

The term “flexible polypeptide linker” or “linker” as used in thecontext of a scFv refers to a peptide linker that consists of aminoacids such as glycine and/or serine residues used alone or incombination, to link variable heavy and variable light chain regionstogether. In one embodiment, the flexible polypeptide linker is aGly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n(SEQ ID NO: 38), where n is a positive integer equal to or greaterthan 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 andn=10 In one embodiment, the flexible polypeptide linkers include, butare not limited to, (Gly4 Ser)4 (SEQ ID NO:27) or (Gly4 Ser)3 (SEQ IDNO:28). In another embodiment, the linkers include multiple repeats of(Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:29). Also included withinthe scope of the invention are linkers described in WO2012/138475,incorporated herein by reference).

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m⁷G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 (SEQ ID NO:30), preferably greater than 64, more preferably greater than 100, mostpreferably greater than 300 or 400. poly(A) sequences can be modifiedchemically or enzymatically to modulate mRNA functionality such aslocalization, stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity and/orduration of a proliferative disorder, or the amelioration of one or moresymptoms (preferably, one or more discernible symptoms) of aproliferative disorder resulting from the administration of one or moretherapies (e.g., one or more therapeutic agents such as a CAR of theinvention). In specific embodiments, the terms “treat”, “treatment” and“treating” refer to the amelioration of at least one measurable physicalparameter of a proliferative disorder, such as growth of a tumor, notnecessarily discernible by the patient. In other embodiments the terms“treat”, “treatment” and “treating”-refer to the inhibition of theprogression of a proliferative disorder, either physically by, e.g.,stabilization of a discernible symptom, physiologically by, e.g.,stabilization of a physical parameter, or both. In other embodiments theterms “treat”, “treatment” and “treating” refer to the reduction orstabilization of tumor size or cancerous cell count.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term, a “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

In the context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder” refers to antigens that are common tospecific hyperproliferative disorders. In certain aspects, thehyperproliferative disorder antigens of the present invention arederived from, cancers including but not limited to primary or metastaticmelanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,non-Hodgkin lymphoma, non-Hodgkin lymphoma, leukemias, uterine cancer,cervical cancer, bladder cancer, kidney cancer and adenocarcinomas suchas breast cancer, prostate cancer, ovarian cancer, pancreatic cancer,and the like.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “specifically binds,” refers to an antibody, or a ligand, whichrecognizes and binds with a cognate binding partner (e.g., a stimulatoryand/or costimulatory molecule present on a T cell) protein present in asample, but which antibody or ligand does not substantially recognize orbind other molecules in the sample.

“Regulatable chimeric antigen receptor (RCAR),” as used herein, refersto a set of polypeptides, typically two in the simplest embodiments,which when in an immune effector cell, provides the cell withspecificity for a target cell, typically a cancer cell, and withregulatable intracellular signal generation. In some embodiments, anRCAR comprises at least an extracellular antigen binding domain, atransmembrane and a cytoplasmic signaling domain (also referred toherein as “an intracellular signaling domain”) comprising a functionalsignaling domain derived from a stimulatory molecule and/orcostimulatory molecule as defined herein in the context of a CARmolecule. In some embodiments, the set of polypeptides in the RCAR arenot contiguous with each other, e.g., are in different polypeptidechains. In some embodiments, the RCAR includes a dimerization switchthat, upon the presence of a dimerization molecule, can couple thepolypeptides to one another, e.g., can couple an antigen binding domainto an intracellular signaling domain. In some embodiments, the RCAR isexpressed in a cell (e.g., an immune effector cell) as described herein,e.g., an RCAR-expressing cell (also referred to herein as “RCARX cell”).In an embodiment the RCARX cell is a T cell, and is referred to as aRCART cell. In an embodiment the RCARX cell is an NK cell, and isreferred to as a RCARN cell. The RCAR can provide the RCAR-expressingcell with specificity for a target cell, typically a cancer cell, andwith regulatable intracellular signal generation or proliferation, whichcan optimize an immune effector property of the RCAR-expressing cell. Inembodiments, an RCAR cell relies at least in part, on an antigen bindingdomain to provide specificity to a target cell that comprises theantigen bound by the antigen binding domain.

“Membrane anchor” or “membrane tethering domain”, as that term is usedherein, refers to a polypeptide or moiety, e.g., a myristoyl group,sufficient to anchor an extracellular or intracellular domain to theplasma membrane.

“Switch domain,” as that term is used herein, e.g., when referring to anRCAR, refers to an entity, typically a polypeptide-based entity, that,in the presence of a dimerization molecule, associates with anotherswitch domain. The association results in a functional coupling of afirst entity linked to, e.g., fused to, a first switch domain, and asecond entity linked to, e.g., fused to, a second switch domain. A firstand second switch domain are collectively referred to as a dimerizationswitch. In embodiments, the first and second switch domains are the sameas one another, e.g., they are polypeptides having the same primaryamino acid sequence, and are referred to collectively as ahomodimerization switch. In embodiments, the first and second switchdomains are different from one another, e.g., they are polypeptideshaving different primary amino acid sequences, and are referred tocollectively as a heterodimerization switch. In embodiments, the switchis intracellular. In embodiments, the switch is extracellular. Inembodiments, the switch domain is a polypeptide-based entity, e.g., FKBPor FRB-based, and the dimerization molecule is small molecule, e.g., arapalogue. In embodiments, the switch domain is a polypeptide-basedentity, e.g., an scFv that binds a myc peptide, and the dimerizationmolecule is a polypeptide, a fragment thereof, or a multimer of apolypeptide, e.g., a myc ligand or multimers of a myc ligand that bindto one or more myc scFvs. In embodiments, the switch domain is apolypeptide-based entity, e.g., myc receptor, and the dimerizationmolecule is an antibody or fragments thereof, e.g., myc antibody.

“Dimerization molecule,” as that term is used herein, e.g., whenreferring to an RCAR, refers to a molecule that promotes the associationof a first switch domain with a second switch domain. In embodiments,the dimerization molecule does not naturally occur in the subject, ordoes not occur in concentrations that would result in significantdimerization. In embodiments, the dimerization molecule is a smallmolecule, e.g., rapamycin or a rapalogue, e.g, RAD001.

The term “bioequivalent” refers to an amount of an agent other than thereference compound (e.g., RAD001), required to produce an effectequivalent to the effect produced by the reference dose or referenceamount of the reference compound (e.g., RAD001). In an embodiment theeffect is the level of mTOR inhibition, e.g., as measured by P70 S6kinase inhibition, e.g., as evaluated in an in vivo or in vitro assay,e.g., as measured by an assay described herein, e.g., the Boulay assay,or measurement of phosphorylated S6 levels by western blot. In anembodiment, the effect is alteration of the ratio of PD-1 positive/PD-1negative immune effector cells, e.g., T cells or NK cells, as measuredby cell sorting. In an embodiment a bioequivalent amount or dose of anmTOR inhibitor is the amount or dose that achieves the same level of P70S6 kinase inhibition as does the reference dose or reference amount of areference compound. In an embodiment, a bioequivalent amount or dose ofan mTOR inhibitor is the amount or dose that achieves the same level ofalteration in the ratio of PD-1 positive/PD-1 negative immune effectorcells, e.g., T cells or NK cells as does the reference dose or referenceamount of a reference compound.

The term “low, immune enhancing, dose” when used in conjunction with anmTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 orrapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTORinhibitor that partially, but not fully, inhibits mTOR activity, e.g.,as measured by the inhibition of P70 S6 kinase activity. Methods forevaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, arediscussed herein. The dose is insufficient to result in complete immunesuppression but is sufficient to enhance the immune response. In anembodiment, the low, immune enhancing, dose of mTOR inhibitor results ina decrease in the number of PD-1 positive immune effector cells, e.g., Tcells or NK cells, and/or an increase in the number of PD-1 negativeimmune effector cells, e.g., T cells or NK cells, or an increase in theratio of PD-1 negative T cells/PD-1 positive immune effector cells,e.g., T cells or NK cells.

In an embodiment, the low, immune enhancing, dose of mTOR inhibitorresults in an increase in the number of naive immune effector cells,e.g., T cells or NK cells. In an embodiment, the low, immune enhancing,dose of mTOR inhibitor results in one or more of the following:

-   -   an increase in the expression of one or more of the following        markers: CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on        memory T cells, e.g., memory T cell precursors;    -   a decrease in the expression of KLRG1, e.g., on memory T cells,        e.g., memory T cell precursors; and    -   an increase in the number of memory T cell precursors, e.g.,        cells with any one or combination of the following        characteristics: increased CD62L^(high), increased CD127^(high),        increased CD27⁺, decreased KLRG1, and increased BCL2;

wherein any of the changes described above occurs, e.g., at leasttransiently, e.g., as compared to a non-treated subject.

“Refractory” as used herein refers to a disease, e.g., cancer, that doesnot respond to a treatment. In embodiments, a refractory cancer can beresistant to a treatment before or at the beginning of the treatment. Inother embodiments, the refractory cancer can become resistant during atreatment. A refractory cancer is also called a resistant cancer.

“Relapsed” or “relapse” as used herein refers to the return orreappearance of a disease (e.g., cancer) or the signs and symptoms of adisease such as cancer after a period of improvement or responsiveness,e.g., after prior treatment of a therapy, e.g., cancer therapy. Theinitial period of responsiveness may involve the level of cancer cellsfalling below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%,2%, or 1%. The reappearance may involve the level of cancer cells risingabove a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or1%. For example, e.g., in the context of B-ALL, the reappearance mayinvolve, e.g., a reappearance of blasts in the blood, bone marrow (>5%),or any extramedullary site, after a complete response. A completeresponse, in this context, may involve <5% BM blast. More generally, inan embodiment, a response (e.g., complete response or partial response)can involve the absence of detectable MRD (minimal residual disease). Inan embodiment, the initial period of responsiveness lasts at least 1, 2,3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4,6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

In some embodiments, a therapy that includes a CD19 inhibitor, e.g., aCD19 CAR therapy, may relapse or be refractory to treatment. The relapseor resistance can be caused by CD19 loss (e.g., an antigen lossmutation) or other CD19 alteration that reduces the level of CD19 (e.g.,caused by clonal selection of CD19-negative clones). A cancer thatharbors such CD19 loss or alteration is referred to herein as a“CD19-negative cancer” or a “CD19-negative relapsed cancer”). It shallbe understood that a CD19-negative cancer need not have 100% loss ofCD19, but a sufficient reduction to reduce the effectiveness of a CD19therapy such that the cancer relapses or becomes refractory. In someembodiments, a CD19-negative cancer results from a CD19 CAR therapy.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

Description

Provided herein are compositions of matter and methods of use for thetreatment or prevention of a disease such as cancer using CD123 chimericantigen receptors (CAR).

In one aspect, the invention provides a number of chimeric antigenreceptors (CAR) comprising an antibody or antibody fragment engineeredfor specific binding to a CD123 protein or fragments thereof. In oneaspect, the invention provides a cell (e.g., an immune effector cell,e.g., a T cell or a NK cell) engineered to express a CAR, wherein theCAR-expressing cell (e.g., “CART” or CAR-expressing NK cell) exhibits anantitumor property. In one aspect a cell is transformed with the CAR andthe at least part of the CAR is expressed on the cell surface. In someembodiments, the cell (e.g., immune effector cell, e.g., T cell or NKcell) is transduced with a viral vector encoding a CAR. In someembodiments, the viral vector is a retroviral vector. In someembodiments, the viral vector is a lentiviral vector. In some suchembodiments, the cell may stably express the CAR. In another embodiment,the cell (e.g., immune effector cell, e.g., T cell or NK cell) istransfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR.In some such embodiments, the cell may transiently express the CAR.

In one aspect, the CD123 binding domain, e.g., the human or humanizedCD123 binding domain, of the CAR is a scFv antibody fragment. In oneaspect, such antibody fragments are functional in that they retain theequivalent binding affinity, e.g., they bind the same antigen withcomparable efficacy, as the IgG antibody having the same heavy and lightchain variable regions. In one aspect such antibody fragments arefunctional in that they provide a biological response that can include,but is not limited to, activation of an immune response, inhibition ofsignal-transduction origination from its target antigen, inhibition ofkinase activity, and the like, as will be understood by a skilledartisan.

In some aspects, the antibodies of the invention are incorporated into achimeric antigen receptor (CAR). In one aspect, the CAR comprises thepolypeptide sequence provided herein as SEQ ID NOS: 98-101, and 125-156.

In one aspect, the CD123 binding domain, e.g., humanized or human CD123binding domain, portion of a CAR of the invention is encoded by atransgene whose sequence has been codon optimized for expression in amammalian cell. In one aspect, entire CAR construct of the invention isencoded by a transgene whose entire sequence has been codon optimizedfor expression in a mammalian cell. Codon optimization refers to thediscovery that the frequency of occurrence of synonymous codons (i.e.,codons that code for the same amino acid) in coding DNA is biased indifferent species. Such codon degeneracy allows an identical polypeptideto be encoded by a variety of nucleotide sequences. A variety of codonoptimization methods is known in the art, and include, e.g., methodsdisclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

In one aspect, the antigen binding domain of the CAR comprises a humanCD123 antibody or antibody fragment. In one aspect, the antigen bindingdomain of the CAR comprises a humanized CD123 antibody or antibodyfragment. In one aspect, the antigen binding domain of the CAR compriseshuman CD123 antibody fragment comprising an scFv. In one aspect, theantigen binding domain of the CAR is a human CD123 scFv. In one aspect,the antigen binding domain of the CAR comprises a humanized CD123antibody fragment comprising an scFv. In one aspect, the antigen bindingdomain of the CAR is a humanized CD123 scFv.

In one aspect, the CAR123 binding domain comprises the scFv portionprovided in SEQ ID NO:157-160 and 184-215. In one aspect the scFvportion is human. In one aspect, the human CAR123 binding domaincomprises the scFv portion provided in SEQ ID NO:157-160. In one aspect,the human CD123 binding domain comprises the scFv portion provided inSEQ ID NO: 157. In one aspect, the human CD123 binding domain comprisesthe scFv portion provided in SEQ ID NO: 158. In one aspect, the humanCD123 binding domain comprises the scFv portion provided in SEQ ID NO:159. In one aspect, the human CD123 binding domain comprises the scFvportion provided in SEQ ID NO: 160. In one aspect, the human CD123binding domain comprises the scFv portion provided in SEQ ID NO: 478. Inone aspect, the human CD123 binding domain comprises the scFv portionprovided in SEQ ID NO: 480. In one aspect, the human CD123 bindingdomain comprises the scFv portion provided in SEQ ID NO: 483. In oneaspect, the human CD123 binding domain comprises the scFv portionprovided in SEQ ID NO: 485.

In one aspect the scFv portion is humanized. In one aspect, thehumanized CAR123 binding domain comprises the scFv portion provided inSEQ ID NO:184-215. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 184. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 185. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 186. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 187. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 188. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 189. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 190. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 191. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 192. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 193. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 194. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 195. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 196. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 197. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 198. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 199. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 200. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 201. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 202. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 203. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 204. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 205. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 206. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 207. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 208. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 209. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 210. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 211. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 212. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 213. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NO: 214. In one aspect,the humanized CD123 binding domain comprises the scFv portion providedin SEQ ID NO: 215. In one aspect, the humanized CD123 binding domaincomprises the scFv portion provided in SEQ ID NOs: 556-587.

Furthermore, the present invention provides CD123 CAR compositions andtheir use in medicaments or methods for treating, among other diseases,cancer or any malignancy or autoimmune diseases involving cells ortissues which express CD123.

In one aspect, the CAR of the invention can be used to eradicateCD123-expressing normal cells, thereby applicable for use as a cellularconditioning therapy prior to cell transplantation. In one aspect, theCD123-expressing normal cell is a CD123-expressing expressing myeloidprogenitor cell and the cell transplantation is a stem celltransplantation.

In one aspect, the invention provides a cell (e.g., an immune effectorcell, e.g., a T cell or NK cell) engineered to express a chimericantigen receptor (e.g., CAR-expressing immune effector cell, e.g., CARTor CAR-expressing NK cell) of the present invention, wherein the cell(e.g., “CART”) exhibits an antitumor property. Accordingly, theinvention provides a CD123-CAR that comprises a CD123 binding domain andis engineered into an immune effector cell, e.g., a T cell or a NK cell,and methods of their use for adoptive therapy.

In one aspect, the CD123-CAR comprises at least one intracellulardomain, e.g., described herein, e.g., selected from the group of a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3zeta signaldomain, and any combination thereof. In one aspect, the CD123-CARcomprises at least one intracellular signaling domain is from one ormore co-stimulatory molecule(s) other than a CD137 (4-1BB) or CD28.

Chimeric Antigen Receptor (CAR)

The present invention encompasses a recombinant DNA construct comprisingsequences encoding a CAR, wherein the CAR comprises an antigen bindingdomain (e.g., antibody, antibody fragment) that binds specifically toCD123 or a fragment thereof, e.g., human CD123, wherein the sequence ofthe CD123 binding domain (e.g., antibody or antibody fragment) is, e.g.,contiguous with and in the same reading frame as a nucleic acid sequenceencoding an intracellular signaling domain. The intracellular signalingdomain can comprise a costimulatory signaling domain and/or a primarysignaling domain, e.g., a zeta chain. The costimulatory signaling domainrefers to a portion of the CAR comprising at least a portion of theintracellular domain of a costimulatory molecule.

In specific aspects, a CAR construct of the invention comprises a scFvdomain selected from the group consisting of SEQ ID NOS:157-160,184-215, 478, 480, 483, 485, and 556-587 wherein the scFv may bepreceded by an optional leader sequence such as provided in SEQ ID NO:1, and followed by an optional hinge sequence such as provided in SEQ IDNO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5, a transmembraneregion such as provided in SEQ ID NO:6, an intracellular signallingdomain that includes SEQ ID NO:7 or SEQ ID NO:8 and a CD3 zeta sequencethat includes SEQ ID NO:9 or SEQ ID NO:10, e.g., wherein the domains arecontiguous with and in the same reading frame to form a single fusionprotein. In some embodiments, the scFv domain is a human scFv domainselected from the group consisting of SEQ ID NOS: 157-160, 478, 480,483, and 485. In some embodiments, the scFv domain is a humanized scFvdomain selected from the group consisting of SEQ ID NOS: 184-215 and556-587. Also included in the invention is a nucleotide sequence thatencodes the polypeptide of each of the scFv fragments selected from thegroup consisting of SEQ ID NO: 157-160, 184-215, 478, 480, 483, 485, and556-587. Also included in the invention is a nucleotide sequence thatencodes the polypeptide of each of the scFv fragments selected from thegroup consisting of SEQ ID NO: 157-160, 184-215, 478, 480, 483, 485, and556-587, and each of the domains of SEQ ID NOS: 1,2, and 6-9, plus theencoded CD123 CAR of the invention.

In one aspect an exemplary CD123CAR constructs comprise an optionalleader sequence, an extracellular antigen binding domain, a hinge, atransmembrane domain, and an intracellular stimulatory domain. In oneaspect an exemplary CD123CAR construct comprises an optional leadersequence, an extracellular antigen binding domain, a hinge, atransmembrane domain, an intracellular costimulatory domain and anintracellular stimulatory domain.

In some embodiments, full-length CD123 CAR sequences are also providedherein as SEQ ID NOS: 98-101 and 125-156, as shown in Table 2 or 6.

An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplaryhinge/spacer sequence is provided as SEQ ID NO:2 or SEQ ID NO:3 or SEQID NO:4 or SEQ ID NO:5. An exemplary transmembrane domain sequence isprovided as SEQ ID NO:6. An exemplary sequence of the intracellularsignaling domain of the 4-1BB protein is provided as SEQ ID NO: 7. Anexemplary sequence of the intracellular signaling domain of CD27 isprovided as SEQ ID NO:8. An exemplary CD3zeta domain sequence isprovided as SEQ ID NO: 9 or SEQ ID NO:10. An exemplary sequence of theintracellular signaling domain of CD28 is provided as SEQ ID NO:43. Anexemplary sequence of the intracellular signaling domain of ICOS isprovided as SEQ ID NO:45.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises the nucleic acid sequenceencoding a CD123 binding domain, e.g., described herein, e.g., that iscontiguous with and in the same reading frame as a nucleic acid sequenceencoding an intracellular signaling domain. In one aspect, a CD123binding domain is selected from one or more of SEQ ID NOS: 157-160,184-215, 478, 480, 483, 485, and 556-587. In some embodiments, the CD123binding domain is a human CD123 binding domain selected from the groupconsisting of SEQ ID NOS: 157-160, 478, 480, 483, and 485. In someembodiments, the CD123 binding domain is a humanized CD123 bindingdomain selected from the group consisting of SEQ ID NOS: 184-215 and556-587. In one aspect, the CD123 binding domain is SEQ ID NO: 157. Inone aspect, the CD123 binding domain is SEQ ID NO: 158. In one aspect,the CD123 binding domain is SEQ ID NO: 159. In one aspect, the CD123binding domain is SEQ ID NO: 160. In one aspect, the CD123 bindingdomain is SEQ ID NO: 184. In one aspect, the CD123 binding domain is SEQID NO: 185. In one aspect, the CD123 binding domain is SEQ ID NO: 186.In one aspect, the CD123 binding domain is SEQ ID NO: 187. In oneaspect, the CD123 binding domain is SEQ ID NO: 188. In one aspect, theCD123 binding domain is SEQ ID NO: 189. In one aspect, the CD123 bindingdomain is SEQ ID NO: 190. In one aspect, the CD123 binding domain is SEQID NO: 191. In one aspect, the CD123 binding domain is SEQ ID NO: 192.In one aspect, the CD123 binding domain is SEQ ID NO: 193. In oneaspect, the CD123 binding domain is SEQ ID NO: 194. In one aspect, theCD123 binding domain is SEQ ID NO: 195. In one aspect, the CD123 bindingdomain is SEQ ID NO: 196. In one aspect, the CD123 binding domain is SEQID NO: 197. In one aspect, the CD123 binding domain is SEQ ID NO: 198.In one aspect, the CD123 binding domain is SEQ ID NO: 199. In oneaspect, the CD123 binding domain is SEQ ID NO: 200. In one aspect, theCD123 binding domain is SEQ ID NO: 201. In one aspect, the CD123 bindingdomain is SEQ ID NO: 202. In one aspect, the CD123 binding domain is SEQID NO: 203. In one aspect, the CD123 binding domain is SEQ ID NO: 204.In one aspect, the CD123 binding domain is SEQ ID NO: 205. In oneaspect, the CD123 binding domain is SEQ ID NO: 206. In one aspect, theCD123 binding domain is SEQ ID NO: 207. In one aspect, the CD123 bindingdomain is SEQ ID NO: 208. In one aspect, the CD123 binding domain is SEQID NO: 209. In one aspect, the CD123 binding domain is SEQ ID NO: 210.In one aspect, the CD123 binding domain is SEQ ID NO: 211. In oneaspect, the CD123 binding domain is SEQ ID NO: 212. In one aspect, theCD123 binding domain is SEQ ID NO: 213. In one aspect, the CD123 bindingdomain is SEQ ID NO: 213. In one aspect, the CD123 binding domain is SEQID NO: 215. In one aspect, the CD123 binding domain is SEQ ID NO: 478.In one aspect, the CD123 binding domain is SEQ ID NO: 480. In oneaspect, the CD123 binding domain is SEQ ID NO: 483. In one aspect, theCD123 binding domain is SEQ ID NO: 485.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises a nucleic acid sequenceencoding a CD123 binding domain, e.g., wherein the sequence iscontiguous with and in the same reading frame as the nucleic acidsequence encoding an intracellular signaling domain. An exemplaryintracellular signaling domain that can be used in the CAR includes, butis not limited to, one or more intracellular signaling domains of, e.g.,CD3-zeta, CD28, 4-1BB, ICOS, and the like. In some instances, the CARcan comprise any combination of CD3-zeta, CD28, 4-1BB, ICOS, and thelike.

In one aspect, the nucleic acid sequence of a CAR construct of theinvention is selected from one or more of SEQ ID NOS:39-42 and 66-97. Inone aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:39. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:40.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:41. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:42.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:66. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:67.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:68. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:69.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:70. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:71.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:72. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:73.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:74. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:75.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:76. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:77.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:78. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:79.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:80. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:81.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:82. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:83.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:84. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:85.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:86. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:87.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:88. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:89.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:90. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:91.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:92. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:93.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:94. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:95.In one aspect, the nucleic acid sequence of a CAR construct comprises(e.g., consists of) SEQ ID NO:96. In one aspect, the nucleic acidsequence of a CAR construct comprises (e.g., consists of) SEQ ID NO:97.The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the nucleic acid of interest can beproduced synthetically, rather than cloned.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.

The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the nucleic acid to beexpressed, and a polyA tail, typically 50-2000 bases in length (SEQ IDNO:35). RNA so produced can efficiently transfect different kinds ofcells. In one embodiment, the template includes sequences for the CAR.In an embodiment, an RNA CAR vector is transduced into a T cell byelectroporation.

Antigen Binding Domain

In one aspect, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state.

In one aspect, the CAR-mediated T-cell response can be directed to anantigen of interest by way of engineering an antigen binding domain thatspecifically binds a desired antigen into the CAR.

In one aspect, the portion of the CAR comprising the antigen bindingdomain comprises an antigen binding domain that targets CD123 or afragment thereof. In one aspect, the antigen binding domain targetshuman CD123 or a fragment thereof.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as antigen binding domain, such as a recombinantfibronectin domain, and the like. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise human or humanized residues for the antigen binding domainof an antibody or antibody fragment.

In some instances, it is beneficial for the antigen binding domain to bederived from the same species in which the CAR will ultimately be usedin. For example, for use in humans, it may be beneficial for the antigenbinding domain of the CAR to comprise human or humanized residues forthe antigen binding domain of an antibody or antibody fragment. Thus, inone aspect, the antigen binding domain comprises a human antibody or anantibody fragment. In one embodiment, the human CD123 binding domaincomprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a human CD123 binding domain described herein, and/or oneor more (e.g., all three) heavy chain complementary determining region 1(HC CDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a humanCD123 binding domain described herein, e.g., a human CD123 bindingdomain comprising one or more, e.g., all three, LC CDRs and one or more,e.g., all three, HC CDRs. In one embodiment, the human CD123 bindingdomain comprises one or more (e.g., all three) heavy chain complementarydetermining region 1 (HC CDR1), heavy chain complementary determiningregion 2 (HC CDR2), and heavy chain complementary determining region 3(HC CDR3) of a human CD123 binding domain described herein, e.g., thehuman CD123 binding domain has two variable heavy chain regions, eachcomprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In oneembodiment, the human CD123 binding domain comprises a human light chainvariable region described herein (e.g., in Table 2 or 9) and/or a humanheavy chain variable region described herein (e.g., in Table 2 or 9). Inone embodiment, the human CD123 binding domain comprises a human heavychain variable region described herein (e.g., in Table 2 or 9), e.g., atleast two human heavy chain variable regions described herein (e.g., inTable 2 or 9). In one embodiment, the CD123 binding domain is a scFvcomprising a light chain and a heavy chain of an amino acid sequence ofTable 2 or 9. In an embodiment, the CD123 binding domain (e.g., an scFv)comprises: a light chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions) of an amino acid sequence of a light chain variableregion provided in Table 2 or 9, or a sequence with 95-99% identity withan amino acid sequence of Table 2; and/or a heavy chain variable regioncomprising an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a heavychain variable region provided in Table 2 or 9, or a sequence with95-99% identity to an amino acid sequence of Table 2 or 9. In oneembodiment, the human CD123 binding domain comprises a sequence selectedfrom a group consisting of SEQ ID NO:157-160, 478, 480, 483, and 485, ora sequence with 95-99% identity thereof. In one embodiment, the humanCD123 binding domain is a scFv, and a light chain variable regioncomprising an amino acid sequence described herein, e.g., in Table 2 or9, is attached to a heavy chain variable region comprising an amino acidsequence described herein, e.g., in Table 2, via a linker, e.g., alinker described herein. In one embodiment, the human CD123 bindingdomain includes a (Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6,preferably 3 or 4 (SEQ ID NO:26). The light chain variable region andheavy chain variable region of a scFv can be, e.g., in any of thefollowing orientations: light chain variable region-linker-heavy chainvariable region or heavy chain variable region-linker-light chainvariable region.

In some aspects, a non-human antibody is humanized, where specificsequences or regions of the antibody are modified to increase similarityto an antibody naturally produced in a human or fragment thereof. Thus,in one aspect, the antigen binding domain comprises a humanized antibodyor an antibody fragment. In one embodiment, the humanized CD123 bindingdomain comprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a humanized CD123 binding domain described herein, and/orone or more (e.g., all three) heavy chain complementary determiningregion 1 (HC CDR1), heavy chain complementary determining region 2 (HCCDR2), and heavy chain complementary determining region 3 (HC CDR3) of ahumanized CD123 binding domain described herein, e.g., a humanized CD123binding domain comprising one or more, e.g., all three, LC CDRs and oneor more, e.g., all three, HC CDRs. In one embodiment, the humanizedCD123 binding domain comprises one or more (e.g., all three) heavy chaincomplementary determining region 1 (HC CDR1), heavy chain complementarydetermining region 2 (HC CDR2), and heavy chain complementarydetermining region 3 (HC CDR3) of a humanized CD123 binding domaindescribed herein, e.g., the humanized CD123 binding domain has twovariable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and aHC CDR3 described herein. In one embodiment, the humanized CD123 bindingdomain comprises a humanized light chain variable region describedherein (e.g., in Table 6) and/or a humanized heavy chain variable regiondescribed herein (e.g., in Table 6). In one embodiment, the humanizedCD123 binding domain comprises a humanized heavy chain variable regiondescribed herein (e.g., in Table 6), e.g., at least two humanized heavychain variable regions described herein (e.g., in Table 6). In oneembodiment, the CD123 binding domain is a scFv comprising a light chainand a heavy chain of an amino acid sequence of Table 6. In anembodiment, the CD123 binding domain (e.g., an scFv) comprises: a lightchain variable region comprising an amino acid sequence having at leastone, two or three modifications (e.g., substitutions) but not more than30, 20 or 10 modifications (e.g., substitutions) of an amino acidsequence of a light chain variable region provided in Table 4, or asequence with 95-99% identity with an amino acid sequence of Table 6;and/or a heavy chain variable region comprising an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions)but not more than 30, 20 or 10 modifications (e.g., substitutions) of anamino acid sequence of a heavy chain variable region provided in Table6, or a sequence with 95-99% identity to an amino acid sequence of Table6. In one embodiment, the humanized CD123 binding domain comprises asequence selected from a group consisting of SEQ ID NO:184-215 and302-333, or a sequence with 95-99% identity thereof. In one embodiment,the humanized CD123 binding domain is a scFv, and a light chain variableregion comprising an amino acid sequence described herein, e.g., inTable 6, is attached to a heavy chain variable region comprising anamino acid sequence described herein, e.g., in Table 6, via a linker,e.g., a linker described herein. In one embodiment, the humanized CD123binding domain includes a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4,5, or 6, preferably 3 or 4 (SEQ ID NO:26). The light chain variableregion and heavy chain variable region of a scFv can be, e.g., in any ofthe following orientations: light chain variable region-linker-heavychain variable region or heavy chain variable region-linker-light chainvariable region.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002),Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods,20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto etal., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., CancerRes., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which isincorporated herein in its entirety by reference. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, for exampleimprove, antigen binding. These framework substitutions are identifiedby methods well-known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized antibody or antibody fragment has one or more amino acidresidues remaining in it from a source which is nonhuman. These nonhumanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. As providedherein, humanized antibodies or antibody fragments comprise one or moreCDRs from nonhuman immunoglobulin molecules and framework regionswherein the amino acid residues comprising the framework are derivedcompletely or mostly from human germline. Multiple techniques forhumanization of antibodies or antibody fragments are well-known in theart and can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized antibodies and antibody fragments, substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a nonhuman species. Humanized antibodies areoften human antibodies in which some CDR residues and possibly someframework (FR) residues are substituted by residues from analogous sitesin rodent antibodies. Humanization of antibodies and antibody fragmentscan also be achieved by veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al.,PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332),the contents of which are incorporated herein by reference herein intheir entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993), the contents ofwhich are incorporated herein by reference herein in their entirety). Insome embodiments, the framework region, e.g., all four frameworkregions, of the heavy chain variable region are derived from a VH4_4-59germline sequence. In one embodiment, the framework region can comprise,one, two, three, four or five modifications, e.g., substitutions, e.g.,from the amino acid at the corresponding murine sequence. In oneembodiment, the framework region, e.g., all four framework regions ofthe light chain variable region are derived from a VK3_1.25 germlinesequence. In one embodiment, the framework region can comprise, one,two, three, four or five modifications, e.g., substitutions, e.g., fromthe amino acid at the corresponding murine sequence.

In some aspects, the portion of a CAR composition of the invention thatcomprises an antibody fragment is humanized with retention of highaffinity for the target antigen and other favorable biologicalproperties. According to one aspect of the invention, humanizedantibodies and antibody fragments are prepared by a process of analysisof the parental sequences and various conceptual humanized productsusing three-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, e.g., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind the target antigen. In this way, FR residues canbe selected and combined from the recipient and import sequences so thatthe desired antibody or antibody fragment characteristic, such asincreased affinity for the target antigen, is achieved. In general, theCDR residues are directly and most substantially involved in influencingantigen binding.

A humanized antibody or antibody fragment may retain a similar antigenicspecificity as the original antibody, e.g., in the present invention,the ability to bind human CD123 or a fragment thereof. In someembodiments, a humanized antibody or antibody fragment may have improvedaffinity and/or specificity of binding to human CD123 or a fragmentthereof.

In one aspect, the antigen binding domain portion comprises one or moresequence selected from SEQ ID NOS:157-160, 184-215, 478, 480, 483, 485,and 556-587. In one aspect, the CD123 CAR that includes a human CD123binding domain is selected from one or more sequence selected from SEQID NOS:157-160, 478, 480, 483, and 485. In one aspect, the CD123 CARthat includes a humanized CD123 binding domain is selected from one ormore sequence selected from SEQ ID NOS:184-215 and 556-587.

In one aspect, the CD123 binding domain is characterized by particularfunctional features or properties of an antibody or antibody fragment.For example, in one aspect, the portion of a CAR composition of theinvention that comprises an antigen binding domain specifically bindshuman CD123 or a fragment thereof. In one aspect, the invention relatesto an antigen binding domain comprising an antibody or antibodyfragment, wherein the antibody binding domain specifically binds to aCD123 protein or fragment thereof, wherein the antibody or antibodyfragment comprises a variable light chain and/or a variable heavy chainthat includes an amino acid sequence of SEQ ID NO: 157-160, 184-215,478, 480, 483, 485, and 556-587. In one aspect, the antigen bindingdomain comprises an amino acid sequence of an scFv selected from SEQ IDNO: 157-160, 184-215, 478, 480, 483, 485, and 556-587. In certainaspects, the scFv is contiguous with and in the same reading frame as aleader sequence. In one aspect the leader sequence is the polypeptidesequence provided as SEQ ID NO:1.

In one aspect, the CD123 binding domain is a fragment, e.g., a singlechain variable fragment (scFv). In one aspect, the CD123 binding domainis a Fv, a Fab, a (Fab′)2, or a bi-functional (e.g. bi-specific) hybridantibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).In one aspect, the antibodies and fragments thereof of the inventionbinds a CD123 protein or a fragment thereof with wild-type or enhancedaffinity.

In some instances, a human scFv can be derived from a display library. Adisplay library is a collection of entities; each entity includes anaccessible polypeptide component and a recoverable component thatencodes or identifies the polypeptide component. The polypeptidecomponent is varied so that different amino acid sequences arerepresented. The polypeptide component can be of any length, e.g. fromthree amino acids to over 300 amino acids. A display library entity caninclude more than one polypeptide component, for example, the twopolypeptide chains of a Fab. In one exemplary embodiment, a displaylibrary can be used to identify a human CD123 binding domain. In aselection, the polypeptide component of each member of the library isprobed with CD123, or a fragment thereof, and if the polypeptidecomponent binds to CD123, the display library member is identified,typically by retention on a support.

Retained display library members are recovered from the support andanalyzed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the polypeptide component, i.e.,the anti-CD123 binding domain, and purification of the polypeptidecomponent for detailed characterization.

A variety of formats can be used for display libraries. Examples includethe phaage display. In phage display, the protein component is typicallycovalently linked to a bacteriophage coat protein. The linkage resultsfrom translation of a nucleic acid encoding the protein component fusedto the coat protein. The linkage can include a flexible peptide linker,a protease site, or an amino acid incorporated as a result ofsuppression of a stop codon. Phage display is described, for example, inU.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20;Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005)Nat Biotechnol. 23(3)344-8. Bacteriophage displaying the proteincomponent can be grown and harvested using standard phage preparatorymethods, e.g. PEG precipitation from growth media. After selection ofindividual display phages, the nucleic acid encoding the selectedprotein components can be isolated from cells infected with the selectedphages or from the phage themselves, after amplification. Individualcolonies or plaques can be picked, the nucleic acid isolated andsequenced.

Other display formats include cell based display (see, e.g., WO03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No.6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) Proc.Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol.18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; andSchaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and E.coli periplasmic display (2005 Nov. 22; PMID: 16337958).

In some instances, scFvs can be prepared according to method known inthe art (see, for example, Bird et al., (1988) Science 242:423-426 andHuston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFvmolecules can be produced by linking VH and VL regions together usingflexible polypeptide linkers. The scFv molecules comprise a linker(e.g., a Ser-Gly linker) with an optimized length and/or amino acidcomposition. The linker length can greatly affect how the variableregions of a scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids) intrachain foldingis prevented. Interchain folding is also required to bring the twovariable regions together to form a functional epitope binding site. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent ApplicationPublication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCTpublication Nos. WO2006/020258 and WO2007/024715, is incorporated hereinby reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreamino acid residues between its VL and VH regions. The linker sequencemay comprise any naturally occurring amino acid. In some embodiments,the linker sequence comprises amino acids glycine and serine. In anotherembodiment, the linker sequence comprises sets of glycine and serinerepeats such as (Gly₄Ser)n, where n is a positive integer equal to orgreater than 1 (SEQ ID NO:25). In one embodiment, the linker can be(Gly₄Ser)₄ (SEQ ID NO:27) or (Gly₄Ser)₃(SEQ ID NO:28). Variation in thelinker length may retain or enhance activity, giving rise to superiorefficacy in activity studies.

Exemplary CD123 CAR Constructs and Antigen Binding Domains

Exemplary CD123 CAR constructs disclose herein comprise an scFv (e.g., ahuman scFv as disclosed in Tables 2, 6 and 9 herein, optionally precededwith an optional leader sequence (e.g., SEQ ID NO:1 and SEQ ID NO:12 forexemplary leader amino acid and nucleotide sequences, respectively). Thesequences of the human scFv fragments (amino acid sequences of SEQ IDNOs:157-160) are provided herein in Table 2. The sequences of human scFvfragments, without the leader sequence, are provided herein in Table 9(SEQ ID NOs: 479, 481, 482, and 484 for the nucleotide sequences, andSEQ ID NOs: 478, 480, 483, and 485 for the amino acid sequences). TheCD123 CAR construct can further include an optional hinge domain, e.g.,a CD8 hinge domain (e.g., including the amino acid sequence of SEQ IDNO: 2 or encoded by a nucleic acid sequence of SEQ ID NO:13); atransmembrane domain, e.g., a CD8 transmembrane domain (e.g., includingthe amino acid sequence of SEQ ID NO: 6 or encoded by the nucleotidesequence of SEQ ID NO: 17); an intracellular domain, e.g., a 4-1BBintracellular domain (e.g., including the amino acid sequence of SEQ IDNO: 7 or encoded by the nucleotide sequence of SEQ ID NO: 18; and afunctional signaling domain, e.g., a CD3 zeta domain (e.g., includingamino acid sequence of SEQ ID NO: 9 or 10, or encoded by the nucleotidesequence of SEQ ID NO: 20 or 21). In certain embodiments, the domainsare contiguous with and in the same reading frame to form a singlefusion protein. In other embodiments, the domain are in separatepolypeptides, e.g., as in an RCAR molecule as described herein.

In certain embodiments, the full length CD123 CAR molecule includes theamino acid sequence of, or is encoded by the nucleotide sequence of,CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3,hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9,hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15,hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21,hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27,hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32, providedin Table 2, 6, or 9, or a sequence substantially (e.g., 95-99%)identical thereto.

In certain embodiments, the CD123 CAR molecule, or the CD123 antigenbinding domain, includes the scFv amino acid sequence of CD123-1,CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4,hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10,hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16,hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22,hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28,hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32, provided in Table 2,6 or 9; or includes the scFv amino acid sequence of, or is encoded bythe nucleotide sequence of, CD123-1, CD123-2, CD123-3, CD123-4,hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6,hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12,hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18,hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24,hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30,hzCD123-31, or hzCD123-32, or a sequence substantially identical (e.g.,95-99% identical, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acidchanges) to any of the aforesaid sequences.

In certain embodiments, the CD123 CAR molecule, or the CD123 antigenbinding domain, includes the heavy chain variable region and/or thelight chain variable region of CD123-1, CD123-2, CD123-3, CD123-4,hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6,hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12,hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18,hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24,hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30,hzCD123-31, or hzCD123-32, provided in Table 2 or 6, or a sequencesubstantially identical (e.g., 95-99% identical, or up to 20, 15, 10, 8,6, 5, 4, 3, 2, or 1 amino acid changes) to any of the aforesaidsequences.

In certain embodiments, the CD123 CAR molecule, or the CD123 antigenbinding domain, includes one, two or three CDRs from the heavy chainvariable region (e.g., HCDR1, HCDR2 and/or HCDR3), provided in Table 3or 7; and/or one, two or three CDRs from the light chain variable region(e.g., LCDR1, LCDR2 and/or LCDR3) of CD123-1, CD123-2, CD123-3, CD123-4,hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6,hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12,hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18,hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24,hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30,hzCD123-31, or hzCD123-32, provided in Table 4 or 8; or a sequencesubstantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or1 amino acid changes) to any of the aforesaid sequences.

In certain embodiments, the CD123 CAR molecule, or the CD123 antigenbinding domain, includes one, two or three CDRs from the heavy chainvariable region (e.g., HCDR1, HCDR2 and/or HCDR3), provided in Table 10;and/or one, two or three CDRs from the light chain variable region(e.g., LCDR1, LCDR2 and/or LCDR3) of CD123-1, CD123-2, CD123-3, CD123-4,hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6,hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12,hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18,hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24,hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30,hzCD123-31, or hzCD123-32, provided in Table 11; or a sequencesubstantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or1 amino acid changes) to any of the aforesaid sequences.

In certain embodiments, the CD123 molecule, or the CD123 antigen bindingdomain, includes one, two or three CDRs from the heavy chain variableregion (e.g., HCDR1, HCDR2 and/or HCDR3), provided in Table 12; and/orone, two or three CDRs from the light chain variable region (e.g.,LCDR1, LCDR2 and/or LCDR3) of CD123-1, CD123-2, CD123-3, CD123-4,hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6,hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12,hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18,hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24,hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30,hzCD123-31, or hzCD123-32, provided in Table 13; or a sequencesubstantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or1 amino acid changes) to any of the aforesaid sequences.

The sequences of CDR sequences of the scFv domains are shown in Tables3, 7, 10, and 12 for the heavy chain variable domains and in Tables 4,8, 11, and 13 for the light chain variable domains. “ID” stands for therespective SEQ ID NO for each CDR.

The CDRs provided in Tables 3, 4, 7, and 8 are according to acombination of the Kabat and Chothia numbering scheme.

TABLE 3 Heavy Chain Variable Domain CDRs Candidate HCDR1 ID HCDR2 IDHCDR3 ID CAR123-2 GYTFTGYYMH 335 WINPNSGGTNYAQKFQG 363 DMNILATVPFDI 391CAR123-3 GYIFTGYYIH 337 WINPNSGGTNYAQKFQG 364 DMNILATVPFDI 392 CAR123-4GYTFTGYYMH 336 WINPNSGGTNYAQKFQG 365 DMNILATVPFDI 393 CAR123-1GYTFTDYYMH 334 WINPNSGDTNYAQKFQG 362 DMNILATVPFDI 390

TABLE 4 Light Chain Variable Domain CDRs Candidate LCDR1 ID LCDR2 IDLCDR3 ID CAR123-2 RASQSISSYLN 419 AAFSLQS 447 QQGDSVPLT 475 CAR123-3RASQSISSYLN 420 AASSLQS 448 QQGDSVPLT 476 CAR123-4 RASQSISSYLN 421AASSLQS 449 QQGDSVPLT 477 CAR123-1 RASQSISTYLN 418 AASSLQS 446 QQGDSVPLT474

TABLE 7 Heavy Chain Variable Domain CDR HCDR1 ID HCDR2 ID HCDR3 IDhzCAR123 GYTFTSYWMN 361 RIDPYDSETHYNQK 389 GNWDDY 417 FKD

TABLE 8 Light Chain Variable Domain CDR LCDR1 ID LCDR2 ID LCDR3 IDhzCAR123 RASKSISKDLA 445 SGSTLQS 473 QQHNKYPYT 47

TABLE 10 Heavy Chain Variable Domain CDRs according to the Kabatnumbering scheme (Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD) Candidate HCDR1 ID HCDR2 ID HCDR3 IDCAR123-2 GYYMH 487 WINPNSGGTNYAQKFQG 492 DMNILATVPFDI 497 CAR123-3 GYYIH488 WINPNSGGTNYAQKFQG 493 DMNILATVPFDI 498 CAR123-4 DYYMH 489WINPNSGDTNYAQKFQG 494 DMNILATVPFDI 499 CAR123-1 GYYMH 486WINPNSGGTNYAQKFQG 491 DMNILATVPFDI 496 hzCAR123-1 SYWMN 490RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-2 SYWMN 490 RIDPYDSETHYNQKFKD495 GNWDDY 500 hzCAR123-3 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500hzCAR123-4 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-5 SYWMN490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-6 SYWMN 490RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-7 SYWMN 490 RIDPYDSETHYNQKFKD495 GNWDDY 500 hzCAR123-8 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500hzCAR123-9 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-10 SYWMN490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-11 SYWMN 490RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-12 SYWMN 490 RIDPYDSETHYNQKFKD495 GNWDDY 500 hzCAR123-13 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500hzCAR123-14 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-15 SYWMN490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-16 SYWMN 490RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-17 SYWMN 490 RIDPYDSETHYNQKFKD495 GNWDDY 500 hzCAR123-18 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500hzCAR123-19 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-20 SYWMN490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-21 SYWMN 490RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-22 SYWMN 490 RIDPYDSETHYNQKFKD495 GNWDDY 500 hzCAR123-23 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500hzCAR123-24 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-25 SYWMN490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-26 SYWMN 490RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-27 SYWMN 490 RIDPYDSETHYNQKFKD495 GNWDDY 500 hzCAR123-28 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500hzCAR123-29 SYWMN 490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-30 SYWMN490 RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-31 SYWMN 490RIDPYDSETHYNQKFKD 495 GNWDDY 500 hzCAR123-32 SYWMN 490 RIDPYDSETHYNQKFKD495 GNWDDY 500

TABLE 11 Light Chain Variable Domain CDRs according to the Kabatnumbering scheme (Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD) Candidate LCDR1 ID LCDR2 ID LCDR3 IDCAR123-2 RASQSISSYLN 502 AASSLQS 507 QQGDSVPLT 512 CAR123-3 RASQSISSYLN503 AASSLQS 508 QQGDSVPLT 513 CAR123-4 RASQSISSYLN 504 AASSLQS 509QQGDSVPLT 514 CAR123-1 RASQSISTYLN 501 AAFSLQS 506 QQGDSVPLT 511hzCAR123-1 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-2RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-3 RASKSISKDLA 505SGSTLQS 510 QQHNKYPYT 515 hzCAR123-4 RASKSISKDLA 505 SGSTLQS 510QQHNKYPYT 515 hzCAR123-5 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515hzCAR123-6 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-7RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-8 RASKSISKDLA 505SGSTLQS 510 QQHNKYPYT 515 hzCAR123-10 RASKSISKDLA 505 SGSTLQS 510QQHNKYPYT 515 hzCAR123-10 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515hzCAR123-11 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-12RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-13 RASKSISKDLA 505SGSTLQS 510 QQHNKYPYT 515 hzCAR123-14 RASKSISKDLA 505 SGSTLQS 510QQHNKYPYT 515 hzCAR123-15 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515hzCAR123-16 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-17RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-18 RASKSISKDLA 505SGSTLQS 510 QQHNKYPYT 515 hzCAR123-19 RASKSISKDLA 505 SGSTLQS 510QQHNKYPYT 515 hzCAR123-20 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515hzCAR123-21 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-22RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-23 RASKSISKDLA 505SGSTLQS 510 QQHNKYPYT 515 hzCAR123-24 RASKSISKDLA 505 SGSTLQS 510QQHNKYPYT 515 hzCAR123-25 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515hzCAR123-26 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-27RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-28 RASKSISKDLA 505SGSTLQS 510 QQHNKYPYT 515 hzCAR123-29 RASKSISKDLA 505 SGSTLQS 510QQHNKYPYT 515 hzCAR123-30 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515hzCAR123-31 RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515 hzCAR123-32RASKSISKDLA 505 SGSTLQS 510 QQHNKYPYT 515

TABLE 12 Heavy Chain Variable Domain CDRs according to the Chothianumbering scheme (Al-Lazikani et al., (1997) JMB 273, 927-948) CandidateHCDR1 ID HCDR2 ID HCDR3 ID CAR123-2 GYTFTGY 517 NPNSGG 522 DMNILATVPFDI527 CAR123-3 GYIFTGY 518 NPNSGG 523 DMNILATVPFDI 528 CAR123-4 GYTFTDY519 NPNSGD 524 DMNILATVPFDI 529 CAR123-1 GYTFTGY 516 NPNSGG 521DMNILATVPFDI 526 hzCAR123-1 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-2GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-3 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-4 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-5GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-6 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-7 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-8GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-9 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-10 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-11GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-12 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-13 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-14GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-15 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-16 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-17GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-18 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-19 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-20GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-21 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-22 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-23GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-24 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-25 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-26GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-27 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-28 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-29GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-30 GYTFTSY 520 DPYDSE 525GNWDDY 530 hzCAR123-31 GYTFTSY 520 DPYDSE 525 GNWDDY 530 hzCAR123-32GYTFTSY 520 DPYDSE 525 GNWDDY 530

TABLE 13 Light Chain Variable Domain CDRs according to the Chothianumbering scheme (Al-Lazikani et al., (1997) JMB 273, 927-948) CandidateLCDR1 ID LCDR2 ID LCDR3 ID CAR123-2 SQSISSY 532 AAS 537 GDSVPL 542CAR123-3 SQSISSY 533 AAS 538 GDSVPL 543 CAR123-4 SQSISSY 534 AAS 539GDSVPL 544 CAR123-1 SQSISTY 531 AAF 536 GDSVPL 541 hzCAR123-1 SKSISKD535 SGS 540 HNKYPY 555 hzCAR123-2 SKSISKD 535 SGS 540 HNKYPY 555hzCAR123-3 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-4 SKSISKD 535 SGS 540HNKYPY 555 hzCAR123-5 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-6 SKSISKD535 SGS 540 HNKYPY 555 hzCAR123-7 SKSISKD 535 SGS 540 HNKYPY 555hzCAR123-8 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-10 SKSISKD 535 SGS540 HNKYPY 555 hzCAR123-10 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-11SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-12 SKSISKD 535 SGS 540 HNKYPY555 hzCAR123-13 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-14 SKSISKD 535SGS 540 HNKYPY 555 hzCAR123-15 SKSISKD 535 SGS 540 HNKYPY 555hzCAR123-16 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-17 SKSISKD 535 SGS540 HNKYPY 555 hzCAR123-18 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-19SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-20 SKSISKD 535 SGS 540 HNKYPY555 hzCAR123-21 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-22 SKSISKD 535SGS 540 HNKYPY 555 hzCAR123-23 SKSISKD 535 SGS 540 HNKYPY 555hzCAR123-24 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-25 SKSISKD 535 SGS540 HNKYPY 555 hzCAR123-26 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-27SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-28 SKSISKD 535 SGS 540 HNKYPY555 hzCAR123-29 SKSISKD 535 SGS 540 HNKYPY 555 hzCAR123-30 SKSISKD 535SGS 540 HNKYPY 555 hzCAR123-31 SKSISKD 535 SGS 540 HNKYPY 555hzCAR123-32 SKSISKD 535 SGS 540 HNKYPY 555

In embodiments, CD123 single chain variable fragments are generated andcloned into lentiviral CAR expression vectors with the intracellularCD3zeta domain and the intracellular co-stimulatory domain of 4-1BB.Names of exemplary fully human CD123 scFvs are depicted in Table 1.Names of exemplary humanized CD123 scFvs are depicted in Table 5.

TABLE 1 CAR-CD123 constructs Construct ID CAR Nickname EBB-C1357-F11CAR123-1 EBB-C1358-B10 CAR123-2 EBB-C1358-D5 CAR123-3 EBB-C1357-C4CAR123-4

TABLE 5 CAR-CD123 constructs Construct ID CAR Nickname VH1_1-46_X_VK1_L8hzCAR-1 VH1_1-46_X_VK3_L6 hzCAR-2 VH1_1-46_X_VK6_A14 hzCAR-3VH1_1-46_X_VK4_B3 hzCAR-4 VK1_L8_X_VH1_1-46 hzCAR-5 VK3_L6_X_VH1_1-46hzCAR-6 VK6_A14_X_VH1_1-46 hzCAR-7 VK4_B3_X_VH1_1-46 hzCAR-8VH7_7-4.1_X_VK1_L8 hzCAR-9 VH7_7-4.1_X_VK3_L6 hzCAR-10VH7_7-4.1_X_VK6_A14 hzCAR-11 VH7_7-4.1_X_VK4_B3 hzCAR-12VK1_L8_X_VH7_7-4.1 hzCAR-13 VK3_L6_X_VH7_7-4.1 hzCAR-14VK6_A14_X_VH7_7-4.1 hzCAR-15 VK4_B3_X_VH7_7-4.1 hzCAR-16VH5_5-A_X_VK1_L8 hzCAR-17 VH5_5-A_X_VK3_L6 hzCAR-18 VH5_5-A_X_VK6_A14hzCAR-19 VH5_5-A_X_VK4_B3 hzCAR-20 VK1_L8_X_VH5_5-A hzCAR-21VK3_L6_X_VH5_5-A hzCAR-22 VK6_A14_X_VH5_5-A hzCAR-23 VK4_B3_X_VH5_5-AhzCAR-24 VH3_3-74_X_VK1_L8 hzCAR-25 VH3_3-74_X_VK3_L6 hzCAR-26VH3_3-74_X_VK6_A14 hzCAR-27 VH3_3-74_X_VK4_B3 hzCAR-28 VK1_L8_X_VH3_3-74hzCAR-29 VK3_L6_X_VH3_3-74 hzCAR-30 VK6_A14_X_VH3_3-74 hzCAR-31VK4_B3_X_VH3_3-74 hzCAR-32

In embodiments, the order in which the VL and VH domains appear in thescFv is varied (i.e., VL-VH, or VH-VL orientation), and where eitherthree or four copies of the “G4S” (SEQ ID NO:25) subunit, in which eachsubunit comprises the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S)₃ (SEQID NO:28) or (G4S)₄(SEQ ID NO:27)), connect the variable domains tocreate the entirety of the scFv domain, as shown in Table 2, Table 6,and Table 9.

The amino acid and nucleic acid sequences of the CD123 scFv domains andCD123 CAR molecules are provided in Table 2, Table 6, and Table 9. Theamino acid sequences for the variable heavy chain and variable lightchain for each scFv is also provided in Table 2 and Table 6. It is notedthat the scFv fragments (SEQ ID NOs: 157-160, and 184-215) with a leadersequence (e.g., the amino acid sequence of SEQ ID NO: 1 or thenucleotide sequence of SEQ ID NO: 12) and without a leader sequence (SEQID NOs: 478, 480, 483, 485, and 556-587) are also encompassed by thepresent invention.

Leader (amino acid sequence) (SEQ ID NO: 1) MALPVTALLLPLALLLHAARP Leader(nucleic acid sequence) (SEQ ID NO: 12)ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC ATGCCGCTAGACCC CD8hinge (amino acid sequence) (SEQ ID NO: 2)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8 hinge (nucleic acidsequence) (SEQ ID NO: 13)ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT CD8 transmembrane (amino acidsequence) (SEQ ID NO: 6) IYIWAPLAGTCGVLLLSLVITLYC CD8 transmembrane(nucleic acid sequence) (SEQ ID NO: 17)ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 4-1BB Intracellular domain (amino acid sequence)(SEQ ID NO: 7) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BBIntracellular domain (nucleic acid sequence) (SEQ ID NO: 18)AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD28 Intracellular domain (amino acidsequence) (SEQ ID NO: 43) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQID NO: 43) CD28 Intracellular domain (nucleotide sequence) (SEQ ID NO:44) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 44) ICOS Intracellular domain(amino acid sequence) (SEQ ID NO: 45) T K K K Y S S S V H D P N G E Y MF M R A V N T A K K S R L T D V T L (SEQ ID NO: 45) ICOS Intracellulardomain (nucleotide sequence) (SEQ ID NO: 46)ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT GACCCTA (SEQ ID NO:46) CD3 zeta domain (amino acid sequence) (SEQ ID NO: 9)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR CD3zeta (nucleic acid sequence) (SEQ ID NO: 20)AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CD3 zeta domain (amino acidsequence; NCBI Reference Sequence NM_000734.3) (SEQ ID NO: 10)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR CD3zeta (nucleic acid sequence; NCBI Reference Sequence NM_000734.3); (SEQID NO: 21) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC IgG4 Hinge (amino acidsequence) (SEQ ID NO: 36)ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge (nucleotide sequence) (SEQID NO: 7) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAA GATG

In embodiments, these clones in Table 2 and 6 all contained a Q/Kresidue change in the signal domain of the co-stimulatory domain derivedfrom CD3zeta chain.

TABLE 2 Exemplary CD123 CAR sequences SEQ Name ID Sequence CAR123-2 40atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccac NTgccgctcggccccaagtgcaactcgtccaaagcggagcggaagtcaagaaacccggagcgagcgtgaaagtgtcctgcaaagcctccggctacacctttacgggctactacatgcactgggtgcgccaggcaccaggacagggtcttgaatggatgggatggatcaaccctaattcgggcggaactaactacgcacagaagttccaggggagagtgactctgactcgggatacctccatctcaactgtctacatggaactctcccgcttgcggtcagatgatacggcagtgtactactgcgcccgcgacatgaatatcctggctaccgtgccgttcgacatctggggacaggggactatggttactgtctcatcgggcggtggaggttcaggaggaggcggctcgggaggcggaggttcggacattcagatgacccagtccccatcctctctgtcggccagcgtcggagatagggtgaccattacctgtcgggcctcgcaaagcatctcctcgtacctcaactggtatcagcaaaagccgggaaaggcgcctaagctgctgatctacgccgcttcgagcttgcaaagcggggtgccatccagattctcgggatcaggctcaggaaccgacttcaccctgaccgtgaacagcctccagccggaggactttgccacttactactgccagcagggagactccgtgccgcttactttcggggggggtacccgcctggagatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagecgcagcgcagatgetccagectacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR123-2 99MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFT AAGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNILATVPFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTFGGGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR123-2 158MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFT scFvGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNILATVPFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTF GGGTRLEIK CAR123-2217 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWI VHNPNSGGTNYAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNI LATVPFDIWGQGTMVTVSSCAR123-2 276 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA VLSSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTFGGGT RLEIK CAR123-3 41atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccac NTgccgctcggccccaagtccaactcgttcaatccggcgcagaagtcaagaagccaggagcatcagtgaaagtgtcctgcaaagcctcaggctacatcttcacgggatactacatccactgggtgcgccaggctccgggccagggccttgagtggatgggctggatcaaccctaactctgggggaaccaactacgctcagaagttccaggggagggtcactatgactcgcgatacctccatctccactgcgtacatggaactctcgggactgagatccgacgatcctgccgtgtactactgcgcccgggacatgaacatcttggcgaccgtgccgtttgacatttggggacagggcaccctcgtcactgtgtcgagcggtggaggaggctcggggggtggcggatcaggagggggaggaagcgacatccagctgactcagagcccatcgtcgttgtccgcgtcggtgggggatagagtgaccattacttgccgcgccagccagagcatctcatcatatctgaattggtaccagcagaagcccggaaaggccccaaaactgctgatctacgctgcaagcagcctccaatcgggagtgccgtcacggttctccgggtccggttcgggaactgactttaccctgaccgtgaattcgctgcaaccggaggatttcgccacgtactactgtcagcaaggagactccgtgccgctgaccttcggtggaggcaccaaggtcgaaatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagecgcagcgcagatgetccagectacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR123-3 100MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYIFT AAGYYIHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDPAVYYCARDMNILATVPFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR123-3 159MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYIFT scFvGYYIHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDPAVYYCARDMNILATVPFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTF GGGTKVEIK CAR123-3218 QVQLVQSGAEVKKPGASVKVSCKASGYIFTGYYIHWVRQAPGQGLEWMGWI VHNPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDPAVYYCARDMNI LATVPFDIWGQGTLVTVSSCAR123-3 277 DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA VLSSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTFGGGT KVEIK CAR123-4 42atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccac NTgccgctcggccccaagtccaactccaacagtcaggcgcagaagtgaaaaagagcggtgcatcggtgaaagtgtcatgcaaagcctcgggctacaccttcactgactactatatgcactggctgcggcaggcaccgggacagggacttgagtggatgggatggatcaacccgaattcaggggacactaactacgcgcagaagttccaggggagagtgaccctgacgagggacacctcaatttcgaccgtctacatggaattgtcgcgcctgagatcggacgatactgctgtgtactactgtgcccgcgacatgaacatcctcgcgactgtgccttttgatatctggggacaggggactatggtcaccgtttcctccgcttccggtggcggaggctcgggaggccgggcctccggtggaggaggcagcgacatccagatgactcagagcccttcctcgctgagcgcctcagtgggagatcgcgtgaccatcacttgccgggccagccagtccatttcgtcctacctcaattggtaccagcagaagccgggaaaggcgcccaagctcttgatctacgctgcgagctccctgcaaagcggggtgccgagccgattctcgggttccggctcgggaaccgacttcactctgaccatctcatccctgcaaccagaggactttgccacctactactgccaacaaggagattctgtcccactgacgttcggcggaggaaccaaggtcgaaatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagecgcagcgcagatgetccagectacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR123-4 101MALPVTALLLPLALLLHAARPQVQLQQSGAEVKKSGASVKVSCKASGYTFT AADYYMHWLRQAPGQGLEWMGWINPNSGDTNYAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNILATVPFDIWGQGTMVTVSSASGGGGSGGRASGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDSVPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK CAR123-4 160MALPVTALLLPLALLLHAARPQVQLQQSGAEVKKSGASVKVSCKASGYTFT scFvDYYMHWLRQAPGQGLEWMGWINPNSGDTNYAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNILATVPFDIWGQGTMVTVSSASGGGGSGGRASGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDSVPL TFGGGTKVEIK CAR123-4219 QVQLQQSGAEVKKSGASVKVSCKASGYTFTDYYMHWLRQAPGQGLEWMGWI VHNPNSGDTNYAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNI LATVPFDIWGQGTMVTVSSCAR123-4 278 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA VLSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDSVPLTFGGGT KVEIK CAR123-1 39atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccac NTgccgctcggccccaagtccaactcgtccagtcaggagcggaagtcaagaagcccggagcgtcagtcaaagtgtcatgcaaagcctcgggctacactttcactgggtactacatgcactgggtgcgccaggctccaggacagggactggaatggatgggatggatcaacccgaactccggtggcaccaattacgcccagaagttccaggggagggtgaccatgactcgcgacacgtcgatcagcaccgcatacatggagctgtcaagactccggtccgacgatactgccgtgtactactgcgcacgggacatgaacattctggccaccgtgccttttgacatctggggtcagggaactatggttaccgtgtcctctggtggaggcggctccggcggggggggaagcggaggcggtggaagcgacattcagatgacccagtcgccttcatccctttcggcgagcgtgggagatcgcgtcactatcacttgtcgggcctcgcagtccatctccacctacctcaattggtaccagcagaagccaggaaaagcaccgaatctgctgatctacgccgcgttttccttgcaatcgggagtgccaagcagattcagcggatcgggatcaggcactgatttcaccctcaccatcaactcgctgcaaccggaggatttcgctacgtactattgccaacaaggagacagcgtgccgctcaccttcggcggagggactaagctggaaatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagecgcagcgcagatgetccagectacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR123-1 98malpvtalllplalllhaarpqvqlvqsgaevkkpgasvkvsckasgytft AAgyymhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardmnilatvpfdiwgqgtmvtvssggggsggggsggggsdiqmtqspsslsasvgdrvtitcrasqsistylnwyqqkpgkapnlliyaafslqsgvpsrfsgsgsgtdftltinslqpedfatyycqqgdsvpltfgggtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgeserfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR123-1 157malpvtalllplalllhaarpqvqlvqsgaevkkpgasvkvsckasgytft scFvgyymhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardmnilatvpfdiwgqgtmvtvssggggsggggsggggsdiqmtqspsslsasvgdrvtitcrasqsistylnwyqqkpgkapnlliyaafslqsgvpsrfsgsgsgtdftltinslqpedfatyycqqgdsvpltf gggtkleik CAR123-1216 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWI VHNPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDMNI LATVPFDIWGQGTMVTVSSCAR123-1 275 DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPNLLIYAA VLFSLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQGDSVPLTFGGGT KLEIK

TABLE 6 Humanized CD123 CAR Sequences SEQ Name ID Sequence hzCAR123- 66ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 1 NTCCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 125MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQA 1 AAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR hzCAR123-1184 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQA scFvPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 243QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 1 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 302DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 1 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 67ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 2 NTCCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 126MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ 2 AAAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-2 185MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 244QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 2 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 303EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 2 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 68ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 3 NTCCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 127MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ 3AAAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-3 186MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 245QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 3 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 304DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 3 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 69ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 4 NTCCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 128MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ 4 AAAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-4 187MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 246QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 4 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 305DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 4 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 70ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 5 NTCCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 129MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 5 AAPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-5 188MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK scFvPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 247QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 5 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 306DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 5 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 71ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 6 NTCCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 130MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 6 AAPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-6 189MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK scFvPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 248QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 6 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 307EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 6 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 72ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 7 NTCCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123-7 131MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK AAPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-7 190MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK scFvPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 249QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 7 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 308DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 7 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 73ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 8 NTCCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTCCAGTCGGGAGCCGAAGTCAAGAAGCCCGGCGCTAGCGTGAAAGTGTCCTGCAAAGCCTCCGGGTACACATTCACCTCCTACTGGATGAATTGGGTCAGACAGGCGCCCGGCCAGGGACTCGAGTGGATGGGAAGGATTGATCCTTACGACTCCGAAACCCATTACAACCAGAAGTTCAAGGACCGCGTGACCATGACTGTGGATAAGTCCACTTCCACCGCTTACATGGAGCTGTCCAGCCTGCGCTCCGAGGATACCGCAGTGTACTACTGCGCCCGGGGAAACTGGGACGACTATTGGGGACAGGGAACTACCGTGACCGTGTCAAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 132MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 8 AAPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-8 191MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK scFvPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 250QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 8 VHQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 309DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 8 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 74ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 9 NTCCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 133MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 9 AAAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123-9 192MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 251QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 9 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 310DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 10 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 75ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 10 NTCCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 134MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 10 AAAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 193MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 10APGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 252QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 10 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 311EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 10 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 76ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 11 NTCCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 135MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 11 AAAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 194MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 11APGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 253QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 11 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 312DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 11 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 77ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 12 NTCCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 136MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 12 AAAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 195MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 12APGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 254QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 12 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 313DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 12 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 78ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 13 NTCCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 137MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 13 AAPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 196MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 13PGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 255QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 13 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 314DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 13 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 79ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 14 NTCCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 138MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 14 AAPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 197MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 14PGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 256QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 14 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 315EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 14 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 80ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 15 NTCCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 139MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 15 AAPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 198MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 15PDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 257QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 15 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 316DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 15 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 81ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 16 NTCCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCCAAGTGCAGCTGGTGCAGTCAGGCAGCGAACTGAAGAAGCCCGGAGCCTCCGTCAAAGTGTCCTGCAAAGCCTCGGGATACACCTTCACCTCCTACTGGATGAACTGGGTCCGCCAGGCACCTGGACAGGGGCTGGAGTGGATGGGAAGGATCGATCCCTACGATTCCGAAACCCATTACAATCAGAAGTTCAAGGACCGGTTTGTGTTCTCCGTGGACAAGTCCGTGTCCACCGCCTACCTCCAAATTAGCAGCCTGAAGGCGGAGGATACAGCTGTCTACTACTGCGCTCGCGGAAACTGGGATGACTATTGGGGCCAGGGAACTACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 140MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 16 AAPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 199MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 16PGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 258QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYN 16 VHQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 317DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 16 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 82ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 17 NTCCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 141MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 17 AAMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 200MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 17MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 259EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 17 VHQKFKDHVTISVDKSISTAYLQWS SLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 318DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 17 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 83ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 18 NTCCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 142MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 18 AAMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 201MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 18MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 260EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 18 VHQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 319EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 18 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 84ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 19 NTCCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 143MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 19 AAMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 202MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 19MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 261EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 19 VHQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 320DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 19 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 85ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 20 NTCCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 144MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 20 AAMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 203MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 20MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 262EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 20 VHQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 321DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 20 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 86ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 21 NTCCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 145MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 21 AAPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 204MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 21PGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 263EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 21 VHQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 322DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 21 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 87ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 22 NTCCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 146MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 22 AAPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 205MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 22PGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 264EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 22 VHQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 323EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 22 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 88ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 23 NTCCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 147MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 23 AAPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 206MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 23PDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 265EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 23 VHQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 324DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 23 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 89ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 24 NTCCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTCAAGAAGCCTGGAGAATCCCTGAGGATCAGCTGCAAAGGCAGCGGGTATACCTTCACCTCCTACTGGATGAATTGGGTCCGCCAGATGCCCGGAAAAGGCCTGGAGTGGATGGGACGGATTGACCCCTACGACTCGGAAACCCATTACAACCAGAAGTTCAAGGATCACGTGACCATCTCCGTGGACAAGTCCATTTCCACTGCGTACCTCCAGTGGTCAAGCCTGAAGGCCTCCGACACTGCTATGTACTACTGCGCACGCGGAAACTGGGATGATTACTGGGGACAGGGAACAACCGTGACTGTGTCCTCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 148MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 24 AAPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 207MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 24PGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 266EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYN 24 VHQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 325DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 24 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 90ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 25 NTCCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 149MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 25 AAAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 208MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 25APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 267EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYN 25 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 326DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 25 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 91ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 26 NTCCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 150MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 26 AAAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 209MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 26APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 268EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYN 26 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 327EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 26 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 92ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 27 NTCCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 151MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 27 AAAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 210MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 27APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 269EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYD SETHYN 27 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 328DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 27 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 93ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 28 NTCCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 152MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 28 AAAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 211MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 28APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 270EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYD SETHYN 28 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 329DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 28 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 94ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 29 NTCCGACGTGCAGCTCACCCAGTCGCCCTCATTTCTGTCGGCCTCAGTGGGAGACAGAGTGACCATTACTTGTCGGGCCTCCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCGCCTAAGTTGCTCATCTACTCGGGGTCGACCCTGCAATCTGGCGTGCCGTCCCGGTTCTCCGGTTCGGGAAGCGGTACCGAATTCACCCTTACTATCTCCTCCCTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGCACAACAAGTACCCGTACACTTTCGGGGGTGGCACGAAGGTCGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 153MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 29 AAPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 212MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 29PGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 271EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYN 29 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 330DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSR 29 VLFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 95ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 30 NTCCGAAGTGGTGCTGACCCAGTCGCCCGCAACCCTCTCTCTGTCGCCGGGAGAACGCGCCACTCTTTCCTGTCGGGCGTCCAAGAGCATCTCAAAGGACCTCGCCTGGTACCAGCAGAAGCCTGGTCAAGCCCCGCGGCTGCTGATCTACTCCGGCTCCACGCTGCAATCAGGAATCCCAGCCAGATTTTCCGGTTCGGGGTCGGGGACTGACTTCACCTTGACCATTAGCTCGCTGGAACCTGAGGACTTCGCCGTGTATTACTGCCAGCAGCACAACAAGTACCCGTACACCTTCGGAGGCGGTACTAAGGTCGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 154MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 30 AAPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 213MALPVTALLLPLALLLHAARPEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 30PGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 272EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYN 30 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 331EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPAR 30 VLFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 96ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 31 NTCCGACGTCGTGATGACCCAGTCACCGGCATTCCTGTCCGTGACTCCCGGAGAAAAGGTCACGATTACTTGCCGGGCGTCCAAGAGCATCTCCAAGGACCTCGCCTGGTACCAACAGAAGCCGGACCAGGCCCCTAAGCTGTTGATCTACTCGGGGTCCACCCTTCAATCGGGAGTGCCATCGCGGTTTAGCGGTTCGGGTTCTGGGACCGACTTCACTTTCACCATCTCCTCACTGGAAGCCGAGGATGCCGCCACTTACTACTGTCAGCAGCACAACAAGTATCCGTACACCTTCGGAGGCGGTACCAAAGTGGAGATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 155MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 31 AAPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 214MALPVTALLLPLALLLHAARPDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 31PDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 273EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYN 31 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 332DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSR 31 VLFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 97ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGC 32 NTCCGACGTGGTCATGACTCAGTCCCCGGACTCACTCGCGGTGTCGCTTGGAGAGAGAGCGACCATCAACTGTCGGGCCTCAAAGAGCATCAGCAAGGACCTGGCCTGGTACCAGCAGAAGCCGGGACAGCCGCCAAAGCTGCTGATCTACTCCGGGTCCACCTTGCAATCTGGTGTCCCTGACCGGTTCTCCGGTTCCGGGTCGGGTACCGACTTCACGCTCACTATTTCGTCGCTGCAAGCCGAAGATGTGGCCGTGTACTATTGCCAACAGCACAACAAGTACCCCTACACTTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGGGGTGGCGGTAGCGGAGGAGGGGGCTCCGGCGGCGGCGGCTCAGGGGGCGGAGGAAGCGAAGTGCAGCTCGTCGAGAGCGGAGGGGGACTGGTGCAGCCCGGAGGAAGCCTGAGGCTGTCCTGCGCTGCCTCCGGCTACACCTTCACCTCCTACTGGATGAACTGGGTCAGACAGGCACCTGGAAAGGGACTGGTCTGGGTGTCGCGCATTGACCCCTACGACTCCGAAACCCATTACAATCAGAAATTCAAGGACCGCTTCACCATCTCCGTGGACAAAGCCAAGAGCACCGCGTACCTCCAAATGAACTCCCTGCGCGCTGAGGATACAGCAGTGTACTATTGCGCCCGGGGAAACTGGGATGATTACTGGGGCCAGGGAACTACTGTGACTGTGTCATCCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg hzCAR123- 156MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 32 AAPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRhzCAR123- 215MALPVTALLLPLALLLHAARPDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 32PGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 274EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYN 32 VHQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 333DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDR 32 VLFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK

In embodiments, a CAR molecule described herein comprises a scFv thatspecifically binds to CD123, and does not contain a leader sequence,e.g., the amino acid sequence SEQ ID NO: 1. Table 9 below provides aminoacid and nucleotide sequences for CD123 scFv sequences that do notcontain a leader sequence SEQ ID NO: 1.

TABLE 9 CD123 CAR scFv sequences SEQ Name ID Sequence CAR123-2 479CAAGTGCAACTCGTCCAAAGCGGAGCGGAAGTCAAGAAACCCGGAGCGAGCGTGAAAGTG scFv - NTTCCTGCAAAGCCTCCGGCTACACCTTTACGGGCTACTACATGCACTGGGTGCGCCAGGCACCAGGACAGGGTCTTGAATGGATGGGATGGATCAACCCTAATTCGGGCGGAACTAACTACGCACAGAAGTTCCAGGGGAGAGTGACTCTGACTCGGGATACCTCCATCTCAACTGTCTACATGGAACTCTCCCGCTTGCGGTCAGATGATACGGCAGTGTACTACTGCGCCCGCGACATGAATATCCTGGCTACCGTGCCGTTCGACATCTGGGGACAGGGGACTATGGTTACTGTCTCATCGGGCGGTGGAGGTTCAGGAGGAGGCGGCTCGGGAGGCGGAGGTTCGGACATTCAGATGACCCAGTCCCCATCCTCTCTGTCGGCCAGCGTCGGAGATAGGGTGACCATTACCTGTCGGGCCTCGCAAAGCATCTCCTCGTACCTCAACTGGTATCAGCAAAAGCCGGGAAAGGCGCCTAAGCTGCTGATCTACGCCGCTTCGAGCTTGCAAAGCGGGGTGCCATCCAGATTCTCGGGATCAGGCTCAGGAACCGACTTCACCCTGACCGTGAACAGCCTCCAGCCGGAGGACTTTGCCACTTACTACTGCCAGCAGGGAGACTCCGTGCCGCTTACTTTCGGGGGGGGTACCCGCCTG GAGATCAAGCAR123-2 480QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY scFv - AAAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNILATVPFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTFGGGTRL EIKCAR123-2 481atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggccccaagtgcaactcgtccaaagcgORF-freegagcggaagtcaagaaacccggagcgagcgtgaaagtgtcctgcaaagcctccggctacacctttacgggctactacatgcactNTgggtgcgccaggcaccaggacagggtcttgaatggatgggatggatcaaccctaattcgggcggaactaactacgcacagaagttccaggggagagtgactctgactcgggatacctccatctcaactgtctacatggaactctcccgcttgcggtcagatgatacggcagtgtactactgcgcccgcgacatgaatatcctggctaccgtgccgttcgacatctggggacaggggactatggttactgtctcatcgggcggtggaggttcaggaggaggcggctcgggaggcggaggttcggacattcagatgacccagtccccatcctctctgtcggccagcgtcggagatagggtgaccattacctgtcgggcctcgcaaagcatctcctcgtacctcaactggtatcagcaaaagccgggaaaggcgcctaagctgctgatctacgccgcttcgagcttgcaaagcggggtgccatccagattctcgggatcaggctcaggaaccgacttcaccctgaccgtgaacagcctccagccggaggactttgccacttactactgccagcagggagactccgtgccgcttactttcggggggggtacccgcctggagatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatattaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcttgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagacgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcggtaagtcgacagctcgattcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgctgcgtcgagagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgctgcctcgacgaattcCAR123-3 482CAAGTCCAACTCGTTCAATCCGGCGCAGAAGTCAAGAAGCCAGGAGCATCAGTGAAAGTG scFv - NTTCCTGCAAAGCCTCAGGCTACATCTTCACGGGATACTACATCCACTGGGTGCGCCAGGCTCCGGGCCAGGGCCTTGAGTGGATGGGCTGGATCAACCCTAACTCTGGGGGAACCAACTACGCTCAGAAGTTCCAGGGGAGGGTCACTATGACTCGCGATACCTCCATCTCCACTGCGTACATGGAACTCTCGGGACTGAGATCCGACGATCCTGCCGTGTACTACTGCGCCCGGGACATGAACATCTTGGCGACCGTGCCGTTTGACATTTGGGGACAGGGCACCCTCGTCACTGTGTCGAGCGGTGGAGGAGGCTCGGGGGGTGGCGGATCAGGAGGGGGAGGAAGCGACATCCAGCTGACTCAGAGCCCATCGTCGTTGTCCGCGTCGGTGGGGGATAGAGTGACCATTACTTGCCGCGCCAGCCAGAGCATCTCATCATATCTGAATTGGTACCAGCAGAAGCCCGGAAAGGCCCCAAAACTGCTGATCTACGCTGCAAGCAGCCTCCAATCGGGAGTGCCGTCACGGTTCTCCGGGTCCGGTTCGGGAACTGACTTTACCCTGACCGTGAATTCGCTGCAACCGGAGGATTTCGCCACGTACTACTGTCAGCAAGGAGACTCCGTGCCGCTGACCTTCGGTGGAGGCACCAAGGTC GAAATCAAGCAR123-3 483QVQLVQSGAEVKKPGASVKVSCKASGYIFTGYYIHWVRQAPGQGLEWMGWINPNSGGTNY scFv - AAAQKFQGRVTMTRDTSISTAYMELSGLRSDDPAVYYCARDMNILATVPFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTVNSLQPEDFATYYCQQGDSVPLTFGGGTKV EIKCAR123-4 484CAAGTCCAACTCCAACAGTCAGGCGCAGAAGTGAAAAAGAGCGGTGCATCGGTGAAAGTG scFv - NTTCATGCAAAGCCTCGGGCTACACCTTCACTGACTACTATATGCACTGGCTGCGGCAGGCACCGGGACAGGGACTTGAGTGGATGGGATGGATCAACCCGAATTCAGGGGACACTAACTACGCGCAGAAGTTCCAGGGGAGAGTGACCCTGACGAGGGACACCTCAATTTCGACCGTCTACATGGAATTGTCGCGCCTGAGATCGGACGATACTGCTGTGTACTACTGTGCCCGCGACATGAACATCCTCGCGACTGTGCCTTTTGATATCTGGGGACAGGGGACTATGGTCACCGTTTCCTCCGCTTCCGGTGGCGGAGGCTCGGGAGGCCGGGCCTCCGGTGGAGGAGGCAGCGACATCCAGATGACTCAGAGCCCTTCCTCGCTGAGCGCCTCAGTGGGAGATCGCGTGACCATCACTTGCCGGGCCAGCCAGTCCATTTCGTCCTACCTCAATTGGTACCAGCAGAAGCCGGGAAAGGCGCCCAAGCTCTTGATCTACGCTGCGAGCTCCCTGCAAAGCGGGGTGCCGAGCCGATTCTCGGGTTCCGGCTCGGGAACCGACTTCACTCTGACCATCTCATCCCTGCAACCAGAGGACTTTGCCACCTACTACTGCCAACAAGGAGATTCTGTCCCACTGACGTTCGGCGGAGGAACCAAGGTCGAAATCAAG CAR123-4 485QVQLQQSGAEVKKSGASVKVSCKASGYTFTDYYMHWLRQAPGQGLEWMGWINPNSGDTNY scFv - AAAQKFQGRVTLTRDTSISTVYMELSRLRSDDTAVYYCARDMNILATVPFDIWGQGTMVTVSSASGGGGSGGRASGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDSVPLTFGGGT KVEIKCAR123-1 478QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY scFv - AAAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDMNILATVPFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPNLLIYAAFSLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQGDSVPLTFGGGTKL EIKhzCAR123- 556QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHY 1 scFvNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVE IKhzCAR123- 557 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ 2 scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 558QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ 3 scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 559QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQ 4 scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 560DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 5 scFvPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 561EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 6 scFvPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 562DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 7 scFvPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 563DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 8 scFvPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 564QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 9 scFvAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 565QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 10APGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 566QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 11APGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 567QVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQ 12APGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 568DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 13PGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 569EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 14PGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 570DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 15PDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 571DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 16PGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 572EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 17MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 573EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 18MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTEGGGTKVEIK hzCAR123- 574EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 19MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAELSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTEGGGTKVEIK hzCAR123- 575EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQ 20MPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTEGGGTKVEIK hzCAR123- 576DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 21PGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 577EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 22PGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 578DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 23PDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 579DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 24PGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSS hzCAR123- 580EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 25APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVQLTQSPSELSASVGDRVTITCRASKSISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTEGGGTKVEIK hzCAR123- 581EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 26APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTEGGGTKVEIK hzCAR123- 582EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 27APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPAELSVTPGEKVTITCRASKSISKDLAWYQQKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTEGGGTKVEIK hzCAR123- 583EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQ 28APGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARG scFvNWDDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQKPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFGGGTKVEIK hzCAR123- 584DVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWYQQK 29PGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 585EVVLTQSPATLSLSPGERATLSCRASKSISKDLAWYQQK 30PGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 586DVVMTQSPAFLSVTPGEKVTITCRASKSISKDLAWYQQK 31PDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS hzCAR123- 587DVVMTQSPDSLAVSLGERATINCRASKSISKDLAWYQQK 32PGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHNKYPYTFG scFvGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSS

In embodiments, the CAR scFv fragments were then cloned into lentiviralvectors to create a full length CAR construct in a single coding frame,and using the EF1 alpha promoter for expression (SEQ ID NO: 11).

EF1 alpha promoterCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA Gly/Ser (SEQ ID NO: 25)GGGGSGly/Ser (SEQ ID NO: 26): This sequence may encompass 1-6 “Gly Gly Gly Gly Ser”repeating units GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS Gly/Ser (SEQ ID NO: 27)GGGGSGGGGS GGGGSGGGGS Gly/Ser (SEQ ID NO: 28) GGGGSGGGGS GGGGSGly/Ser (SEQ ID NO: 29) GGGS PolyA: (A)₅₀₀₀ (SEQ ID NO: 30)This sequence may encompass 50-5000 adenines.PolyA: (T)₁₀₀ (SEQ ID NO: 31) PolyA: (T)₅₀₀₀ (SEQ ID NO: 32)This sequence may encompass 50-5000 thymines.PolyA: (A)₅₀₀₀ (SEQ ID NO: 33)This sequence may encompass 100-5000 adenines.PolyA: (A)₄₀₀ (SEQ ID NO: 34)This sequence may encompass 100-400 adenines.PolyA: (A)₂₀₀₀ (SEQ ID NO: 35)This sequence may encompass 50-2000 adenines.Gly/Ser (SEQ ID NO: 709): This sequence may encompass 1-10 “Gly Gly Gly Ser”repeating units GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS

The CAR scFv fragments can be cloned into lentiviral vectors to create afull length CAR construct in a single coding frame, and using the EF1alpha promoter for expression (SEQ ID NO: 11).

The CAR construct can include a Gly/Ser linker having one or more of thefollowing sequences: GGGGS (SEQ ID NO:25); encompassing 1-6 “Gly Gly GlyGly Ser” repeating units, e.g., GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS (SEQ IDNO:26); GGGGSGGGGS GGGGSGGGGS (SEQ ID NO:27); GGGGSGGGGS GGGGS (SEQ IDNO:28); GGGS (SEQ ID NO:29); or encompassing 1-10 “Gly Gly Gly Ser”repeating units, e.g., GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS (SEQID NO:709). In embodiments, the CAR construct include a poly A sequence,e.g., a sequence encompassing 50-5000 or 100-5000 adenines (e.g., SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35), or a sequenceencompassing 50-5000 thymines (e.g., SEQ ID NO:31, SEQ ID NO:32).Alternatively, the CAR construct can include, for example, a linkerincluding the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 704)

Bispecific CARs

In an embodiment a multispecific antibody molecule is a bispecificantibody molecule. A bispecific antibody has specificity for no morethan two antigens. A bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence which has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence that has binding specificity for a second epitope. In anembodiment the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodimentthe first and second epitopes overlap. In an embodiment the first andsecond epitopes do not overlap. In an embodiment the first and secondepitopes are on different antigens, e.g., different proteins (ordifferent subunits of a multimeric protein). In an embodiment abispecific antibody molecule comprises a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a first epitope and a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a second epitope. In an embodiment a bispecific antibodymolecule comprises a half antibody having binding specificity for afirst epitope and a half antibody having binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a half antibody, or fragment thereof, having bindingspecificity for a first epitope and a half antibody, or fragmentthereof, having binding specificity for a second epitope. In anembodiment a bispecific antibody molecule comprises a scFv, or fragmentthereof, have binding specificity for a first epitope and a scFv, orfragment thereof, have binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a multi-specific (e.g.,a bispecific or a trispecific) antibody molecule. Protocols forgenerating bispecific or heterodimeric antibody molecules are known inthe art; including but not limited to, for example, the “knob in a hole”approach described in, e.g., U.S. Pat. No. 5,731,168; the electrostaticsteering Fc pairing as described in, e.g., WO 09/089004, WO 06/106905and WO 2010/129304; Strand Exchange Engineered Domains (SEED)heterodimer formation as described in, e.g., WO 07/110205; Fab armexchange as described in, e.g., WO 08/119353, WO 2011/131746, and WO2013/060867; double antibody conjugate, e.g., by antibody cross-linkingto generate a bi-specific structure using a heterobifunctional reagenthaving an amine-reactive group and a sulfhydryl reactive group asdescribed in, e.g., U.S. Pat. No. 4,433,059; bispecific antibodydeterminants generated by recombining half antibodies (heavy-light chainpairs or Fabs) from different antibodies through cycle of reduction andoxidation of disulfide bonds between the two heavy chains, as describedin, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., threeFab′ fragments cross-linked through sulfhdryl reactive groups, asdescribed in, e.g., U.S. Pat. No. 5,273,743; biosynthetic bindingproteins, e.g., pair of scFvs cross-linked through C-terminal tailspreferably through disulfide or amine-reactive chemical cross-linking,as described in, e.g., U.S. Pat. No. 5,534,254; bifunctional antibodies,e.g., Fab fragments with different binding specificities dimerizedthrough leucine zippers (e.g., c-fos and c-jun) that have replaced theconstant domain, as described in, e.g., U.S. Pat. No. 5,582,996;bispecific and oligospecific mono- and oligovalent receptors, e.g.,VH-CH1 regions of two antibodies (two Fab fragments) linked through apolypeptide spacer between the CH1 region of one antibody and the VHregion of the other antibody typically with associated light chains, asdescribed in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA-antibodyconjugates, e.g., crosslinking of antibodies or Fab fragments through adouble stranded piece of DNA, as described in, e.g., U.S. Pat. No.5,635,602; bispecific fusion proteins, e.g., an expression constructcontaining two scFvs with a hydrophilic helical peptide linker betweenthem and a full constant region, as described in, e.g., U.S. Pat. No.5,637,481; multivalent and multispecific binding proteins, e.g., dimerof polypeptides having first domain with binding region of Ig heavychain variable region, and second domain with binding region of Ig lightchain variable region, generally termed diabodies (higher orderstructures are also encompassed creating for bispecifc, trispecific, ortetraspecific molecules, as described in, e.g., U.S. Pat. No. 5,837,242;minibody constructs with linked VL and VH chains further connected withpeptide spacers to an antibody hinge region and CH3 region, which can bedimerized to form bispecific/multivalent molecules, as described in,e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a shortpeptide linker (e.g., 5 or 10 amino acids) or no linker at all in eitherorientation, which can form dimers to form bispecific diabodies; trimersand tetramers, as described in, e.g., U.S. Pat. No. 5,844,094; String ofVH domains (or VL domains in family members) connected by peptidelinkages with crosslinkable groups at the C-terminus further associatedwith VL domains to form a series of FVs (or scFvs), as described in,e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptideswith both a VH and a VL domain linked through a peptide linker arecombined into multivalent structures through non-covalent or chemicalcrosslinking to form, e.g., homobivalent, heterobivalent, trivalent, andtetravalent structures using both scFV or diabody type format, asdescribed in, e.g., U.S. Pat. No. 5,869,620. Additional exemplarymultispecific and bispecific molecules and methods of making the sameare found, for example, in U.S. Pat. Nos. 5,910,573, 5,932,448,5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396,6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,185, 6,833,441,7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866, 7,612,181,US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1,US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1,US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1,US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1,US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1,U2006263367A1, US2007004909A1, US2007087381A1, US2007128150A1,US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1,US2008069820A1, US2008152645A1, US2008171855A1, US2008241884A1,US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1,US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1,US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1,US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2, WO04081051A1,WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2,WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1,WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2,WO9964460A1. The contents of the above-referenced applications areincorporated herein by reference in their entireties.

Within each antibody or antibody fragment (e.g., scFv) of a bispecificantibody molecule, the VH can be upstream or downstream of the VL. Insome embodiments, the upstream antibody or antibody fragment (e.g.,scFv) is arranged with its VH (VH₁) upstream of its VL (VL₁) and thedownstream antibody or antibody fragment (e.g., scFv) is arranged withits VL (VL₂) upstream of its VH (VH₂), such that the overall bispecificantibody molecule has the arrangement VH₁-VL₁-VL₂-VH₂. In otherembodiments, the upstream antibody or antibody fragment (e.g., scFv) isarranged with its VL (VL₁) upstream of its VH (VH₁) and the downstreamantibody or antibody fragment (e.g., scFv) is arranged with its VH (VH₂)upstream of its VL (VL₂), such that the overall bispecific antibodymolecule has the arrangement VL₁-VH₁-VH₂-VL₂. Optionally, a linker isdisposed between the two antibodies or antibody fragments (e.g., scFvs),e.g., between VL₁ and VL₂ if the construct is arranged asVH₁-VL₁-VL₂-VH₂, or between VH₁ and VH₂ if the construct is arranged asVL₁-VH₁-VH₂-VL₂. The linker may be a linker as described herein, e.g., a(Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQID NO: 64). In general, the linker between the two scFvs should be longenough to avoid mispairing between the domains of the two scFvs.Optionally, a linker is disposed between the VL and VH of the firstscFv. Optionally, a linker is disposed between the VL and VH of thesecond scFv. In constructs that have multiple linkers, any two or moreof the linkers can be the same or different. Accordingly, in someembodiments, a bispecific CAR comprises VLs, VHs, and optionally one ormore linkers in an arrangement as described herein.

In one aspect, the bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence, e.g., a scFv, which hasbinding specificity for CD123, e.g., comprises a scFv as describedherein, e.g., as described in Table 2, Table 6, or Table 9, or comprisesthe light chain CDRs and/or heavy chain CDRs from a CD123 scFv describedherein, and a second immunoglobulin variable domain sequence that hasbinding specificity for a second epitope on a different antigen. In someaspects the second immunoglobulin variable domain sequence has bindingspecificity for an antigen expressed on AML cells, e.g., an antigenother than CD123. For example, the second immunoglobulin variable domainsequence has binding specificity for CLL-1. As another example, thesecond immunoglobulin variable domain sequence has binding specificityfor CD33. As another example, the second immunoglobulin variable domainsequence has binding specificity for CD34. As another example, thesecond immunoglobulin variable domain sequence has binding specificityfor FLT3. For example, the second immunoglobulin variable domainsequence has binding specificity for folate receptor beta. In someaspects, the second immunoglobulin variable domain sequence has bindingspecificity for an antigen expressed on B-cells, for example, CD19,CD20, CD22 or ROR1.

Chimeric TCR

In one aspect, the CD123 antibodies and antibody fragments of thepresent invention (for example, those disclosed in Tables 2, 6 or 9) canbe grafted to one or more constant domain of a T cell receptor (“TCR”)chain, for example, a TCR alpha or TCR beta chain, to create an chimericTCR that binds specificity to CD123. Without being bound by theory, itis believed that chimeric TCRs will signal through the TCR complex uponantigen binding. For example, a CD123 scFv as disclosed herein, can begrafted to the constant domain, e.g., at least a portion of theextracellular constant domain, the transmembrane domain and thecytoplasmic domain, of a TCR chain, for example, the TCR alpha chainand/or the TCR beta chain. As another example, a CD123 antibodyfragment, for example a VL domain as described herein, can be grafted tothe constant domain of a TCR alpha chain, and a CD123 antibody fragment,for example a VH domain as described herein, can be grafted to theconstant domain of a TCR beta chain (or alternatively, a VL domain maybe grafted to the constant domain of the TCR beta chain and a VH domainmay be grafted to a TCR alpha chain). As another example, the CDRs of aCD123 antibody or antibody fragment, e.g., the CDRs of a CD123 antibodyor antibody fragment as described in Tables 3, 4, 5, 6, 7, 8, 10, 11, 12or 13 may be grafted into a TCR alpha and/or beta chain to create achimeric TCR that binds specifically to CD123. For example, the LCDRsdisclosed herein may be grafted into the variable domain of a TCR alphachain and the HCDRs disclosed herein may be grafted to the variabledomain of a TCR beta chain, or vice versa. Such chimeric TCRs may beproduced by methods known in the art (For example, Willemsen R A et al,Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004;11: 487-496; Aggen et al, Gene Ther. 2012 April; 19(4):365-74).

Stability and Mutations

The stability of a CD123 binding domain, e.g., scFv molecules (e.g.,soluble scFv) can be evaluated in reference to the biophysicalproperties (e.g., thermal stability) of a conventional control scFvmolecule or a full length antibody. In one embodiment, the human scFvhas a thermal stability that is greater than about 0.1, about 0.25,about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees,about 13 degrees, about 14 degrees, or about 15 degrees Celsius than acontrol binding molecule (e.g. a conventional scFv molecule) in thedescribed assays.

The improved thermal stability of the CD123 binding domain, e.g., scFvis subsequently conferred to the entire CART123 construct, leading toimproved therapeutic properties of the CART123 construct. The thermalstability of the CD123 binding domain, e.g., scFv can be improved by atleast about 2° C. or 3° C. as compared to a conventional antibody. Inone embodiment, the CD123 binding domain, e.g., scFv has a 1° C.improved thermal stability as compared to a conventional antibody. Inanother embodiment, the CD123 binding domain, e.g., scFv has a 2° C.improved thermal stability as compared to a conventional antibody. Inanother embodiment, the scFv has a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15° C. improved thermal stability as compared to a conventionalantibody. Comparisons can be made, for example, between the scFvmolecules disclosed herein and full length antibodies. Thermal stabilitycan be measured using methods known in the art. For example, in oneembodiment, Tm can be measured. Methods for measuring Tm and othermethods of determining protein stability are described in more detailbelow.

Mutations in scFv alter the stability of the scFv and improve theoverall stability of the scFv and the CART123 construct. Stability ofthe humanized or human scFv is determined using measurements such as Tm,temperature denaturation and temperature aggregation.

The binding capacity of the mutant scFvs can be determined using assaysdescribed in the Examples.

In one embodiment, the CD123 binding domain, e.g., scFv comprises atleast one mutation such that the mutated scFv confers improved stabilityto the CART123 construct. In another embodiment, the CD123 bindingdomain, e.g., scFv comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mutations arising from the humanization process such that the mutatedscFv confers improved stability to the CART123 construct.

Methods of Evaluating Protein Stability

The stability of an antigen binding domain may be assessed using, e.g.,the methods described below. Such methods allow for the determination ofmultiple thermal unfolding transitions where the least stable domaineither unfolds first or limits the overall stability threshold of amultidomain unit that unfolds cooperatively (e.g., a multidomain proteinwhich exhibits a single unfolding transition). The least stable domaincan be identified in a number of additional ways. Mutagenesis can beperformed to probe which domain limits the overall stability.Additionally, protease resistance of a multidomain protein can beperformed under conditions where the least stable domain is known to beintrinsically unfolded via DSC or other spectroscopic methods (Fontana,et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol.393: 672-692). Once the least stable domain is identified, the sequenceencoding this domain (or a portion thereof) may be employed as a testsequence in the methods.

a) Thermal Stability

The thermal stability of the compositions may be analyzed using a numberof non-limiting biophysical or biochemical techniques known in the art.In certain embodiments, thermal stability is evaluated by analyticalspectroscopy.

An exemplary analytical spectroscopy method is Differential ScanningCalorimetry (DSC). DSC employs a calorimeter which is sensitive to theheat absorbances that accompany the unfolding of most proteins orprotein domains (see, e.g. Sanchez-Ruiz, et al., Biochemistry, 27:1648-52, 1988). To determine the thermal stability of a protein, asample of the protein is inserted into the calorimeter and thetemperature is raised until the Fab or scFv unfolds. The temperature atwhich the protein unfolds is indicative of overall protein stability.

Another exemplary analytical spectroscopy method is Circular Dichroism(CD) spectroscopy. CD spectrometry measures the optical activity of acomposition as a function of increasing temperature. Circular dichroism(CD) spectroscopy measures differences in the absorption of left-handedpolarized light versus right-handed polarized light which arise due tostructural asymmetry. A disordered or unfolded structure results in a CDspectrum very different from that of an ordered or folded structure. TheCD spectrum reflects the sensitivity of the proteins to the denaturingeffects of increasing temperature and is therefore indicative of aprotein's thermal stability (see van Mierlo and Steemsma, J.Biotechnol., 79(3):281-98, 2000).

Another exemplary analytical spectroscopy method for measuring thermalstability is Fluorescence Emission Spectroscopy (see van Mierlo andSteemsma, supra). Yet another exemplary analytical spectroscopy methodfor measuring thermal stability is Nuclear Magnetic Resonance (NMR)spectroscopy (see, e.g. van Mierlo and Steemsma, supra).

The thermal stability of a composition can be measured biochemically. Anexemplary biochemical method for assessing thermal stability is athermal challenge assay. In a “thermal challenge assay”, a compositionis subjected to a range of elevated temperatures for a set period oftime. For example, in one embodiment, test scFv molecules or moleculescomprising scFv molecules are subject to a range of increasingtemperatures, e.g., for 1-1.5 hours. The activity of the protein is thenassayed by a relevant biochemical assay. For example, if the protein isa binding protein (e.g. an scFv or scFv-containing polypeptide) thebinding activity of the binding protein may be determined by afunctional or quantitative ELISA.

Such an assay may be done in a high-throughput format and thosedisclosed in the Examples using E. coli and high throughput screening. Alibrary of CD123 binding domains, e.g., scFv variants may be createdusing methods known in the art. CD123 binding domains, e.g., scFvexpression may be induced and the CD123 binding domains, e.g., scFv maybe subjected to thermal challenge. The challenged test samples may beassayed for binding and those CD123 binding domains, e.g., scFvs whichare stable may be scaled up and further characterized.

Thermal stability is evaluated by measuring the melting temperature (Tm)of a composition using any of the above techniques (e.g. analyticalspectroscopy techniques). The melting temperature is the temperature atthe midpoint of a thermal transition curve wherein 50% of molecules of acomposition are in a folded state (See e.g., Dimasi et al. (2009) J. MolBiol. 393: 672-692). In one embodiment, Tm values for a CD123 bindingdomain, e.g., scFv are about 40° C., 41° C., 42° C., 43° C., 44° C., 45°C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54°C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63°C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72°C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81°C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90°C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99°C., 100° C. In one embodiment, Tm values for an IgG is about 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77°C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86°C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95°C., 96° C., 97° C., 98° C., 99° C., 100° C. In one embodiment, Tm valuesfor an multivalent antibody is about 40° C., 41° C., 42° C., 43° C., 44°C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53°C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62°C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71°C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80°C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89°C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98°C., 99° C., 100° C.

Thermal stability is also evaluated by measuring the specific heat orheat capacity (Cp) of a composition using an analytical calorimetrictechnique (e.g. DSC). The specific heat of a composition is the energy(e.g. in kcal/mol) is required to rise by 1° C., the temperature of 1mol of water. As large Cp is a hallmark of a denatured or inactiveprotein composition. The change in heat capacity (ΔCp) of a compositionis measured by determining the specific heat of a composition before andafter its thermal transition. Thermal stability may also be evaluated bymeasuring or determining other parameters of thermodynamic stabilityincluding Gibbs free energy of unfolding (ΔG), enthalpy of unfolding(ΔH), or entropy of unfolding (ΔS). One or more of the above biochemicalassays (e.g. a thermal challenge assay) are used to determine thetemperature (i.e. the T_(C) value) at which 50% of the compositionretains its activity (e.g. binding activity).

In addition, mutations to the CD123 binding domain, e.g., scFv alter thethermal stability of the CD123 binding domain, e.g., scFv compared withthe unmutated CD123 binding domain, e.g., scFv. When the humanized orhuman CD123 binding domain, e.g., scFv is incorporated into a CART123construct, the CD123 binding domain, e.g., humanized or human scFvconfers thermal stability to the overall CD123 CART construct. In oneembodiment, the CD123 binding domain, e.g., scFv comprises a singlemutation that confers thermal stability to the CD123 binding domain,e.g., scFv. In another embodiment, the CD123 binding domain, e.g., scFvcomprises multiple mutations that confer thermal stability to the CD123binding domain, e.g., scFv. In one embodiment, the multiple mutations inthe CD123 binding domain, e.g., scFv have an additive effect on thermalstability of the CD123 binding domain, e.g., scFv.

b) % Aggregation

The stability of a composition can be determined by measuring itspropensity to aggregate. Aggregation can be measured by a number ofnon-limiting biochemical or biophysical techniques. For example, theaggregation of a composition may be evaluated using chromatography, e.g.Size-Exclusion Chromatography (SEC). SEC separates molecules on thebasis of size. A column is filled with semi-solid beads of a polymericgel that will admit ions and small molecules into their interior but notlarge ones. When a protein composition is applied to the top of thecolumn, the compact folded proteins (i.e. non-aggregated proteins) aredistributed through a larger volume of solvent than is available to thelarge protein aggregates. Consequently, the large aggregates move morerapidly through the column, and in this way the mixture can be separatedor fractionated into its components. Each fraction can be separatelyquantified (e.g. by light scattering) as it elutes from the gel.Accordingly, the % aggregation of a composition can be determined bycomparing the concentration of a fraction with the total concentrationof protein applied to the gel. Stable compositions elute from the columnas essentially a single fraction and appear as essentially a single peakin the elution profile or chromatogram.

c) Binding Affinity

The stability of a composition can be assessed by determining its targetbinding affinity. A wide variety of methods for determining bindingaffinity are known in the art. An exemplary method for determiningbinding affinity employs surface plasmon resonance. Surface plasmonresonance is an optical phenomenon that allows for the analysis ofreal-time biospecific interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627;Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson,B., et al. (1991) Anal. Biochem. 198:268-277.

In one aspect, the antigen binding domain of the CAR comprises an aminoacid sequence that is homologous to an antigen binding domain amino acidsequence described herein, and the antigen binding domain retains thedesired functional properties of the CD123 antibody fragments describedherein. In one specific aspect, the CAR composition of the inventioncomprises an antibody fragment. In a further aspect, that antibodyfragment comprises an scFv.

In various aspects, the antigen binding domain of the CAR is engineeredby modifying one or more amino acids within one or both variable regions(e.g., VH and/or VL), for example within one or more CDR regions and/orwithin one or more framework regions. In one specific aspect, the CARcomposition of the invention comprises an antibody fragment. In afurther aspect, that antibody fragment comprises an scFv.

It will be understood by one of ordinary skill in the art that theantibody or antibody fragment of the invention may further be modifiedsuch that they vary in amino acid sequence (e.g., from wild-type), butnot in desired activity. For example, additional nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made to the protein For example, anonessential amino acid residue in a molecule may be replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members, e.g., a conservative substitution, in which an aminoacid residue is replaced with an amino acid residue having a similarside chain, may be made.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences that are thesame. Two sequences are “substantially identical” if two sequences havea specified percentage of amino acid residues or nucleotides that arethe same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over aspecified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman,(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., (1977) Nuc. AcidsRes. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, (1988)Comput. Appl. Biosci. 4:11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

In one aspect, the present invention contemplates modifications of thestarting antibody or fragment (e.g., scFv) amino acid sequence thatgenerate functionally equivalent molecules. For example, the VH or VL ofa CD123 binding domain, e.g., scFv, comprised in the CAR can be modifiedto retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VLframework region of the CD123 binding domain, e.g., scFv. The presentinvention contemplates modifications of the entire CAR construct, e.g.,modifications in one or more amino acid sequences of the various domainsof the CAR construct in order to generate functionally equivalentmolecules. The CAR construct can be modified to retain at least about70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identity of the starting CAR construct.

Transmembrane Domain

With respect to the transmembrane domain, in various embodiments, a CARcan be designed to comprise a transmembrane domain that is attached tothe extracellular domain of the CAR. A transmembrane domain can includeone or more additional amino acids adjacent to the transmembrane region,e.g., one or more amino acid associated with the extracellular region ofthe protein from which the transmembrane was derived (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region)and/or one or more additional amino acids associated with theintracellular region of the protein from which the transmembrane proteinis derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids ofthe intracellular region). In one aspect, the transmembrane domain isone that is associated with one of the other domains of the CAR is used.In some instances, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membraneproteins, e.g., to minimize interactions with other members of thereceptor complex. In one aspect, the transmembrane domain is capable ofhomodimerization with another CAR on the CAR-expressing cell, e.g., CARTcell, cell surface. In a different aspect the amino acid sequence of thetransmembrane domain may be modified or substituted so as to minimizeinteractions with the binding domains of the native binding partnerpresent in the same CAR-expressing cell, e.g., CART cell.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspectthe transmembrane domain is capable of signaling to the intracellulardomain(s) whenever the CAR has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane region(s) of e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,CD137, CD154. In some embodiments, a transmembrane domain may include atleast the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27,LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR,HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19,IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, PAG/Cbp, NKG2D, NKG2C, and CD19.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the antigen binding domain of theCAR, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human Ig (immunoglobulin) hinge,e.g., an IgG4 hinge, or a CD8a hinge. In one embodiment, the hinge orspacer comprises (e.g., consists of) the amino acid sequence of SEQ IDNO:2. In one aspect, the transmembrane domain comprises (e.g., consistsof) a transmembrane domain of SEQ ID NO: 6.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequenceESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO:3).In some embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence ofGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO:14).

In one aspect, the hinge or spacer comprises an IgD hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLED QREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO:4). Insome embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence ofAGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACG TGACTGACCATT(SEQ ID NO:15).

In one aspect, the transmembrane domain may be recombinant, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. In one aspect a triplet of phenylalanine, tryptophan andvaline can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic region of the CAR. A glycine-serine doublet providesa particularly suitable linker. For example, in one aspect, the linkercomprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:5). In someembodiments, the linker is encoded by a nucleotide sequence ofGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:16).

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Cytoplasmic Domain

The Cytoplasmic Domain or Region of the Present CAR Includes anIntracellular signaling domain. An intracellular signaling domain iscapable of activation of at least one of the normal effector functionsof the immune cell in which the CAR has been introduced.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondaryand/or costimulatory signal is also required. Thus, T cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic domain, e.g., acostimulatory domain).

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, CD278 (also known as “ICOS”), FcεRI, DAP10, DAP12, and CD66d. Inone embodiment, a CAR of the invention comprises an intracellularsignaling domain, e.g., a primary signaling domain of CD3-zeta.

In one embodiment, a primary signaling domain comprises a modified ITAMdomain, e.g., a mutated ITAM domain which has altered (e.g., increasedor decreased) activity as compared to the native ITAM domain. In oneembodiment, a primary signaling domain comprises a modifiedITAM-containing primary intracellular signaling domain, e.g., anoptimized and/or truncated ITAM-containing primary intracellularsignaling domain. In an embodiment, a primary signaling domain comprisesone, two, three, four or more ITAM motifs.

Further examples of molecules containing a primary intracellularsignaling domain that are of particular use in the invention includethose of DAP10, DAP12, and CD32.

The intracellular signalling domain of the CAR can comprise the primarysignalling domain, e.g., CD3-zeta signaling domain, by itself or it canbe combined with any other desired intracellular signaling domain(s)useful in the context of a CAR of the invention. For example, theintracellular signaling domain of the CAR can comprise a primarysignalling domain, e.g., CD3 zeta chain portion, and a costimulatorysignaling domain. The costimulatory signaling domain refers to a portionof the CAR comprising the intracellular domain of a costimulatorymolecule. A costimulatory molecule is a cell surface molecule other thanan antigen receptor or its ligands that is required for an efficientresponse of lymphocytes to an antigen. Examples of such moleculesinclude a MHC class I molecule, TNF receptor proteins,Immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), activating NK cellreceptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28,CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS,ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7,NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, CD19a, and a ligand that specifically binds with CD83. Forexample, CD27 costimulation has been demonstrated to enhance expansion,effector function, and survival of human CART cells in vitro andaugments human T cell persistence and antitumor activity in vivo (Songet al. Blood. 2012; 119(3):696-706).

The intracellular signaling sequences within the cytoplasmic portion ofthe CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling sequence. In one embodiment, a glycine-serine doublet can beused as a suitable linker. In one embodiment, a single amino acid, e.g.,an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28. In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain of4-1BB. In one aspect, the signaling domain of 4-1BB is a signalingdomain of SEQ ID NO: 7. In one aspect, the signaling domain of CD3-zetais a signaling domain of SEQ ID NO: 9 (mutant CD3-zeta) or SEQ ID NO: 10(wild type human CD3-zeta).

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD27. In one aspect, the signaling domain of CD27 comprises an aminoacid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQID NO:8). In one aspect, the signalling domain of CD27 is encoded by anucleic acid sequence ofAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCA GCCTATCGCTCC(SEQ ID NO:19).

In one aspect, the signaling domain of CD27 comprises an amino acidsequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ IDNO:8).

In one aspect, the signalling domain of CD27 is encoded by a nucleicacid sequence of AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCA GCCTATCGCTCC(SEQ ID NO:19).

In one aspect, the intracellular is designed to comprise the signalingdomain of CD3-zeta and the signaling domain of CD28. In one aspect, thesignaling domain of CD28 comprises an amino acid sequence of SEQ ID NO:379. In one aspect, the signaling domain of CD28 is encoded by a nucleicacid sequence of SEQ ID NO: 380.

In one aspect, the intracellular is designed to comprise the signalingdomain of CD3-zeta and the signaling domain of ICOS. In one aspect, thesignaling domain of CD28 comprises an amino acid sequence of SEQ ID NO:381. In one aspect, the signaling domain of ICOS is encoded by a nucleicacid sequence of SEQ ID NO: 382.

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target (CD123) or a differenttarget (e.g., CD19, CD33, CLL-1, CD34, FLT3, or folate receptor beta).In one embodiment, the second CAR includes an antigen binding domain toa target expressed on acute myeloid leukemia cells, such as, e.g., CD19,CD33, CLL-1, CD34, FLT3, or folate receptor beta. In one embodiment, theCAR-expressing cell comprises a first CAR that targets a first antigenand includes an intracellular signaling domain having a costimulatorysignaling domain but not a primary signaling domain, and a second CARthat targets a second, different, antigen and includes an intracellularsignaling domain having a primary signaling domain but not acostimulatory signaling domain. While not wishing to be bound by theory,placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27,ICOS or OX-40, onto the first CAR, and the primary signaling domain,e.g., CD3 zeta, on the second CAR can limit the CAR activity to cellswhere both targets are expressed. In one embodiment, the CAR expressingcell comprises a first CD123 CAR that includes a CD123 binding domain, atransmembrane domain and a costimulatory domain and a second CAR thattargets an antigen other than CD123 (e.g., an antigen expressed on AMLcells, e.g., CD19, CD33, CLL-1, CD34, FLT3, or folate receptor beta) andincludes an antigen binding domain, a transmembrane domain and a primarysignaling domain. In another embodiment, the CAR expressing cellcomprises a first CD123 CAR that includes a CD123 binding domain, atransmembrane domain and a primary signaling domain and a second CARthat targets an antigen other than CD123 (e.g., an antigen expressed onAML cells, e.g., CD19, CD33, CLL-1, CD34, FLT3, or folate receptor beta)and includes an antigen binding domain to the antigen, a transmembranedomain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing cell comprises a CD123 CARdescribed herein and an inhibitory CAR. In one embodiment, theinhibitory CAR comprises an antigen binding domain that binds an antigenfound on normal cells but not cancer cells, e.g., normal cells that alsoexpress CD123. In one embodiment, the inhibitory CAR comprises theantigen binding domain, a transmembrane domain and an intracellulardomain of an inhibitory molecule. For example, the intracellular domainof the inhibitory CAR can be an intracellular domain of PD1, PD-L1,PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5),LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276),B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHCclass II, GAL9, adenosine, and TGFR (e.g., TGFR beta).

In one embodiment, when the CAR-expressing cell comprises two or moredifferent CARs, the antigen binding domains of the different CARs can besuch that the antigen binding domains do not interact with one another.For example, a cell expressing a first and second CAR can have anantigen binding domain of the first CAR, e.g., as a fragment, e.g., anscFv, that does not form an association with the antigen binding domainof the second CAR, e.g., the antigen binding domain of the second CAR isa VHH.

In some embodiments, the antigen binding domain comprises a singledomain antigen binding (SDAB) molecules include molecules whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies. SDAB molecules may be any of the art, or any future singledomain molecules. SDAB molecules may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, lamprey, fish,shark, goat, rabbit, and bovine. This term also includes naturallyoccurring single domain antibody molecules from species other thanCamelidae and sharks.

In one aspect, an SDAB molecule can be derived from a variable region ofthe immunoglobulin found in fish, such as, for example, that which isderived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainmolecules derived from a variable region of NAR (“IgNARs”) are describedin WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, an SDAB molecule is a naturally occurringsingle domain antigen binding molecule known as heavy chain devoid oflight chains. Such single domain molecules are disclosed in WO 9404678and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.For clarity reasons, this variable domain derived from a heavy chainmolecule naturally devoid of light chain is known herein as a VHH ornanobody to distinguish it from the conventional VH of four chainimmunoglobulins. Such a VHH molecule can be derived from Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain moleculesnaturally devoid of light chain; such VHHs are within the scope of theinvention.

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display).

It has also been discovered, that cells having a plurality of chimericmembrane embedded receptors comprising an antigen binding domain thatinteractions between the antigen binding domain of the receptors can beundesirable, e.g., because it inhibits the ability of one or more of theantigen binding domains to bind its cognate antigen. Accordingly,disclosed herein are cells having a first and a second non-naturallyoccurring chimeric membrane embedded receptor comprising antigen bindingdomains that minimize such interactions. Also disclosed herein arenucleic acids encoding a first and a second non-naturally occurringchimeric membrane embedded receptor comprising a antigen binding domainsthat minimize such interactions, as well as methods of making and usingsuch cells and nucleic acids. In an embodiment the antigen bindingdomain of one of said first said second non-naturally occurring chimericmembrane embedded receptor, comprises an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence.

In some embodiments, the claimed invention comprises a first and secondCAR, wherein the antigen binding domain of one of said first CAR saidsecond CAR does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofsaid first CAR said second CAR is an scFv, and the other is not an scFv.In some embodiments, the antigen binding domain of one of said first CARsaid second CAR comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence. In some embodiments, the antigen binding domain of oneof said first CAR said second CAR comprises a nanobody. In someembodiments, the antigen binding domain of one of said first CAR saidsecond CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of said first CARsaid second CAR comprises an scFv, and the other comprises a single VHdomain, e.g., a camelid, shark, or lamprey single VH domain, or a singleVH domain derived from a human or mouse sequence. In some embodiments,the antigen binding domain of one of said first CAR said second CARcomprises an scFv, and the other comprises a nanobody. In someembodiments, the antigen binding domain of one of said first CAR saidsecond CAR comprises an scFv, and the other comprises a camelid VHHdomain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of said first CAR to its cognate antigen isnot substantially reduced by the presence of said second CAR. In someembodiments, binding of the antigen binding domain of said first CAR toits cognate antigen in the presence of said second CAR is 85%, 90%, 95%,96%, 97%, 98% or 99% of binding of the antigen binding domain of saidfirst CAR to its cognate antigen in the absence of said second CAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of said first CAR said second CAR, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of said first CAR said secondCAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99% lessthan if both were scFv antigen binding domains.

In another aspect, the CAR-expressing cell described herein can furtherexpress another agent, e.g., an agent which enhances the activity of aCAR-expressing cell. For example, in one embodiment, the agent can be anagent which inhibits an inhibitory molecule. Inhibitory molecules, e.g.,PD1, can, in some embodiments, decrease the ability of a CAR-expressingcell to mount an immune effector response. Examples of inhibitorymolecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 orCD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR(e.g., TGFR beta). In one embodiment, the agent which inhibits aninhibitory molecule, e.g., is a molecule described herein, e.g., anagent that comprises a first polypeptide, e.g., an inhibitory molecule,associated with a second polypeptide that provides a positive signal tothe cell, e.g., an intracellular signaling domain described herein. Inone embodiment, the agent comprises a first polypeptide, e.g., of aninhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT,LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9,adenosine, and TGFR (e.g., TGFR beta), or a fragment of any of these(e.g., at least a portion of an extracellular domain of any of these),and a second polypeptide which is an intracellular signaling domaindescribed herein (e.g., comprising a costimulatory domain (e.g., 41BB,CD27 or CD28, e.g., as described herein) and/or a primary signalingdomain (e.g., a CD3 zeta signaling domain described herein). In oneembodiment, the agent comprises a first polypeptide of PD1 or a fragmentthereof (e.g., at least a portion of an extracellular domain of PD1),and a second polypeptide of an intracellular signaling domain describedherein (e.g., a CD28 signaling domain described herein and/or a CD3 zetasignaling domain described herein). In embodiments, the CAR-expressingcell described herein comprises a switch costimulatory receptor, e.g.,as described in WO 2013/019615, which is incorporated herein byreference in its entirety. PD1 is an inhibitory member of the CD28family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.PD-1 is expressed on activated B cells, T cells and myeloid cells (Agataet al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2have been shown to downregulate T cell activation upon binding to PD1(Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 NatImmunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 isabundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank etal. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 ClinCancer Res 10:5094). Immune suppression can be reversed by inhibitingthe local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises the extracellular domain (ECD) ofan inhibitory molecule, e.g., Programmed Death 1 (PD1), can be fused toa transmembrane domain and intracellular signaling domains such as 41BBand CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment,the PD1 CAR, when used in combinations with a CD123 CAR describedherein, improves the persistence of the CAR-expressing cell, e.g., Tcell or NK cell. In one embodiment, the CAR is a PD1 CAR comprising theextracellular domain of PD1 indicated as underlined in SEQ ID NO: 24. Inone embodiment, the PD1 CAR comprises the amino acid sequence of SEQ IDNO:24.

(SEQ ID NO: 24) Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the PD1 CAR comprises the amino acid sequenceprovided below (SEQ ID NO:22).

(SEQ ID NO: 22) pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelryterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the agent comprises a nucleic acid sequence encodingthe PD1 CAR, e.g., the PD1 CAR described herein. In one embodiment, thenucleic acid sequence for the PD1 CAR is shown below, with the PD1 ECDunderlined below in SEQ ID NO: 23

(SEQ ID NO: 23) atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc.

In another aspect, the present invention provides a population ofCAR-expressing cells, e.g., CART cells or CAR-expressing NK cells. Insome embodiments, the population of CAR-expressing cells comprises amixture of cells expressing different CARs. For example, in oneembodiment, the population of CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) can include a first cell expressing a CARhaving a CD123 binding domain described herein, and a second cellexpressing a CAR having a different CD123 binding domain, e.g., a CD123binding domain described herein that differs from the CD123 bindingdomain in the CAR expressed by the first cell. As another example, thepopulation of CAR-expressing cells can include a first cell expressing aCAR that includes a CD123 binding domain, e.g., as described herein, anda second cell expressing a CAR that includes an antigen binding domainto a target other than CD123 (e.g., CD33, CD34, CLL-1, FLT3, or folatereceptor beta). In one embodiment, the population of CAR-expressingcells includes, e.g., a first cell expressing a CAR that includes aprimary intracellular signaling domain, and a second cell expressing aCAR that includes a secondary signaling domain, e.g., a costimulatorysignaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having aCD123 domain described herein, and a second cell expressing anotheragent, e.g., an agent which enhances the activity of a CAR-expressingcell. For example, in one embodiment, the agent can be an agent whichinhibits an inhibitory molecule. Inhibitory molecules, e.g., can, insome embodiments, decrease the ability of a CAR-expressing cell to mountan immune effector response. Examples of inhibitory molecules includePD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFR beta). Inone embodiment, the agent which inhibits an inhibitory molecule, e.g.,is a molecule described herein, e.g., an agent that comprises a firstpolypeptide, e.g., an inhibitory molecule, associated with a secondpolypeptide that provides a positive signal to the cell, e.g., anintracellular signaling domain described herein. In one embodiment, theagent comprises a first polypeptide, e.g., of an inhibitory moleculesuch as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80,CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,MHC class I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFR beta),or a fragment of any of these (e.g., at least a portion of anextracellular domain of any of these), and a second polypeptide which isan intracellular signaling domain described herein (e.g., comprising acostimulatory domain (e.g., 41BB, CD27 or CD28, e.g., as describedherein) and/or a primary signaling domain (e.g., a CD3 zeta signalingdomain described herein). In one embodiment, the agent comprises a firstpolypeptide of PD1 or a fragment thereof (e.g., at least a portion ofthe extracellular domain of PD1), and a second polypeptide of anintracellular signaling domain described herein (e.g., a CD28 signalingdomain described herein and/or a CD3 zeta signaling domain describedherein).

In one aspect, the present invention provides methods comprisingadministering a population of CAR-expressing cells, e.g., CART cells orCAR-expressing NK cells, e.g., a mixture of cells expressing differentCARs, in combination with another agent, e.g., a kinase inhibitor, suchas a kinase inhibitor described herein. In another aspect, the presentinvention provides methods comprising administering a population ofcells wherein at least one cell in the population expresses a CAR havingan anti-cancer associated antigen binding domain as described herein,and a second cell expressing another agent, e.g., an agent whichenhances the activity of a CAR-expressing cell, in combination withanother agent, e.g., a kinase inhibitor, such as a kinase inhibitordescribed herein.

Natural Killer Cell Receptor (NKR) CARs

In an embodiment, the CAR molecule described herein comprises one ormore components of a natural killer cell receptor (NKR), thereby formingan NKR-CAR. The NKR component can be a transmembrane domain, a hingedomain, or a cytoplasmic domain from any of the following natural killercell receptors: killer cell immunoglobulin-like receptor (KIR), e.g.,KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2,KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, andKIR3DP1; natural cyotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46;signaling lymphocyte activation molecule (SLAM) family of immune cellreceptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, andCD2F-10; Fc receptor (FcR), e.g., CD16, and CD64; and Ly49 receptors,e.g., LY49A, LY49C. The NKR-CAR molecules described herein may interactwith an adaptor molecule or intracellular signaling domain, e.g., DAP12.Exemplary configurations and sequences of CAR molecules comprising NKRcomponents are described in International Publication No. WO2014/145252,the contents of which are hereby incorporated by reference.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The splitCAR approach is described in more detail in publications WO2014/055442and WO2014/055657, incorporated herein by reference. Briefly, a splitCAR system comprises a cell expressing a first CAR having a firstantigen binding domain and a costimulatory domain (e.g., 41BB), and thecell also expresses a second CAR having a second antigen binding domainand an intracellular signaling domain (e.g., CD3 zeta). When the cellencounters the first antigen, the costimulatory domain is activated, andthe cell proliferates. When the cell encounters the second antigen, theintracellular signaling domain is activated and cell-killing activitybegins. Thus, the CAR-expressing cell is only fully activated in thepresence of both antigens. In embodiments the first antigen bindingdomain recognizes CD123, e.g., comprises an antigen binding domaindescribed herein, and the second antigen binding domain recognizes anantigen expressed on acute myeloid leukemia cells, e.g., CLL-1, CD33,CD34, FLT3, or folate receptor beta. In embodiments the first antigenbinding domain recognizes CD123, e.g., comprises an antigen bindingdomain described herein, and the second antigen binding domainrecognizes an antigen expressed on B-cells, e.g., CD19, CD20, CD22 orROR1.

Strategies for Regulating Chimeric Antigen Receptors

There are many ways CAR activities can be regulated. In someembodiments, a regulatable CAR (RCAR) where the CAR activity canbecontrolled is desirable to optimize the safety and efficacy of a CARtherapy. For example, inducing apoptosis using, e.g., a caspase fused toa dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov.3; 365(18):1673-1683), can be used as a safety switch in the CAR therapyof the instant invention. In another example, CAR-expressing cells canalso express an inducible Caspase-9 (iCaspase-9) molecule that, uponadministration of a dimerizer drug (e.g., rimiducid (also called AP1903(Bellicum Pharmaceuticals) or AP20187 (Ariad)) leads to activation ofthe Caspase-9 and apoptosis of the cells. The iCaspase-9 moleculecontains a chemical inducer of dimerization (CID) binding domain thatmediates dimerization in the presence of a CID. This results ininducible and selective depletion of CAR-expressing cells. In somecases, the iCaspase-9 molecule is encoded by a nucleic acid moleculeseparate from the CAR-encoding vector(s). In some cases, the iCaspase-9molecule is encoded by the same nucleic acid molecule as theCAR-encoding vector. The iCaspase-9 can provide a safety switch to avoidany toxicity of CAR-expressing cells. See, e.g., Song et al. Cancer GeneTher. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963; and DiStasi et al. N. Engl. J. Med. 2011; 365:1673-83.

Alternative strategies for regulating the CAR therapy of the instantinvention include utilizing small molecules or antibodies thatdeactivate or turn off CAR activity, e.g., by deleting CAR-expressingcells, e.g., by inducing antibody dependent cell-mediated cytotoxicity(ADCC). For example, CAR-expressing cells described herein may alsoexpress an antigen that is recognized by molecules capable of inducingcell death, e.g., ADCC or complement-induced cell death. For example,CAR expressing cells described herein may also express a receptorcapable of being targeted by an antibody or antibody fragment. Examplesof such receptors include EpCAM, VEGFR, integrins (e.g., integrins ανβ3,α4, αI¾β3, α4β7, α5β1, ανβ3, αν), members of the TNF receptorsuperfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferonreceptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1,TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD1 1, CD1 1a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25,CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80,CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5,CD319/SLAMF7, and EGFR, and truncated versions thereof (e.g., versionspreserving one or more extracellular epitopes but lacking one or moreregions within the cytoplasmic domain).

For example, a CAR-expressing cell described herein may also express atruncated epidermal growth factor receptor (EGFR) which lacks signalingcapacity but retains the epitope that is recognized by molecules capableof inducing ADCC, e.g., cetuximab (ERBITUX®), such that administrationof cetuximab induces ADCC and subsequent depletion of the CAR-expressingcells (see, e.g., WO2011/056894, and Jonnalagadda et al., Gene Ther.2013; 20(8)853-860). Another strategy includes expressing a highlycompact marker/suicide gene that combines target epitopes from both CD32and CD20 antigens in the CAR-expressing cells described herein, whichbinds rituximab, resulting in selective depletion of the CAR-expressingcells, e.g., by ADCC (see, e.g., Philip et al., Blood. 2014;124(8)1277-1287). Other methods for depleting CAR-expressing cellsdescribed herein include administration of CAMPATH, a monoclonalanti-CD52 antibody that selectively binds and targets maturelymphocytes, e.g., CAR-expressing cells, for destruction, e.g., byinducing ADCC. In other embodiments, the CAR-expressing cell can beselectively targeted using a CAR ligand, e.g., an anti-idiotypicantibody. In some embodiments, the anti-idiotypic antibody can causeeffector cell activity, e.g, ADCC or ADC activities, thereby reducingthe number of CAR-expressing cells. In other embodiments, the CARligand, e.g., the anti-idiotypic antibody, can be coupled to an agentthat induces cell killing, e.g., a toxin, thereby reducing the number ofCAR-expressing cells. Alternatively, the CAR molecules themselves can beconfigured such that the activity can be regulated, e.g., turned on andoff, as described below.

In other embodiments, a CAR-expressing cell described herein may alsoexpress a target protein recognized by the T cell depleting agent. Inone embodiment, the target protein is CD20 and the T cell depletingagent is an anti-CD20 antibody, e.g., rituximab. In such embodiment, theT cell depleting agent is administered once it is desirable to reduce oreliminate the CAR-expressing cell, e.g., to mitigate the CAR inducedtoxicity. In other embodiments, the T cell depleting agent is ananti-CD52 antibody, e.g., alemtuzumab, as described in the Examplesherein.

In other embodiments, a RCAR comprises a set of polypeptides, typicallytwo in the simplest embodiments, in which the components of a standardCAR described herein, e.g., an antigen binding domain and anintracellular signaling domain, are partitioned on separate polypeptidesor members. In some embodiments, the set of polypeptides include adimerization switch that, upon the presence of a dimerization molecule,can couple the polypeptides to one another, e.g., can couple an antigenbinding domain to an intracellular signaling domain. Additionaldescription and exemplary configurations of such regulatable CARs areprovided herein and in International Publication No. WO 2015/090229,hereby incorporated by reference in its entirety.

In an aspect, an RCAR comprises two polypeptides or members: 1) anintracellular signaling member comprising an intracellular signalingdomain, e.g., a primary intracellular signaling domain described herein,and a first switch domain; 2) an antigen binding member comprising anantigen binding domain, e.g., that specifically binds a tumor antigendescribed herein, as described herein and a second switch domain.Optionally, the RCAR comprises a transmembrane domain described herein.In an embodiment, a transmembrane domain can be disposed on theintracellular signaling member, on the antigen binding member, or onboth. (Unless otherwise indicated, when members or elements of an RCARare described herein, the order can be as provided, but other orders areincluded as well. In other words, in an embodiment, the order is as setout in the text, but in other embodiments, the order can be different.E.g., the order of elements on one side of a transmembrane region can bedifferent from the example, e.g., the placement of a switch domainrelative to a intracellular signaling domain can be different, e.g.,reversed).

In an embodiment, the first and second switch domains can form anintracellular or an extracellular dimerization switch. In an embodiment,the dimerization switch can be a homodimerization switch, e.g., wherethe first and second switch domain are the same, or a heterodimerizationswitch, e.g., where the first and second switch domain are differentfrom one another.

In embodiments, an RCAR can comprise a “multi switch.” A multi switchcan comprise heterodimerization switch domains or homodimerizationswitch domains. A multi switch comprises a plurality of, e.g., 2, 3, 4,5, 6, 7, 8, 9, or 10, switch domains, independently, on a first member,e.g., an antigen binding member, and a second member, e.g., anintracellular signaling member. In an embodiment, the first member cancomprise a plurality of first switch domains, e.g., FKBP-based switchdomains, and the second member can comprise a plurality of second switchdomains, e.g., FRB-based switch domains. In an embodiment, the firstmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain, and the secondmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain.

In an embodiment, the intracellular signaling member comprises one ormore intracellular signaling domains, e.g., a primary intracellularsignaling domain and one or more costimulatory signaling domains.

In an embodiment, the antigen binding member may comprise one or moreintracellular signaling domains, e.g., one or more costimulatorysignaling domains. In an embodiment, the antigen binding membercomprises a plurality, e.g., 2 or 3 costimulatory signaling domainsdescribed herein, e.g., selected from 4-1BB, CD28, CD27, ICOS, and OX40,and in embodiments, no primary intracellular signaling domain. In anembodiment, the antigen binding member comprises the followingcostimulatory signaling domains, from the extracellular to intracellulardirection: 4-1BB-CD27; 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB;OX40-CD28; CD28-OX40; CD28-4-1BB; or 4-1BB-CD28. In such embodiments,the intracellular binding member comprises a CD3zeta domain. In one suchembodiment the RCAR comprises (1) an antigen binding member comprising,an antigen binding domain, a transmembrane domain, and two costimulatorydomains and a first switch domain; and (2) an intracellular signalingdomain comprising a transmembrane domain or membrane tethering domainand at least one primary intracellular signaling domain, and a secondswitch domain.

An embodiment provides RCARs wherein the antigen binding member is nottethered to the surface of the CAR cell. This allows a cell having anintracellular signaling member to be conveniently paired with one ormore antigen binding domains, without transforming the cell with asequence that encodes the antigen binding member. In such embodiments,the RCAR comprises: 1) an intracellular signaling member comprising: afirst switch domain, a transmembrane domain, an intracellular signalingdomain, e.g., a primary intracellular signaling domain, and a firstswitch domain; and 2) an antigen binding member comprising: an antigenbinding domain, and a second switch domain, wherein the antigen bindingmember does not comprise a transmembrane domain or membrane tetheringdomain, and, optionally, does not comprise an intracellular signalingdomain. In some embodiments, the RCAR may further comprise 3) a secondantigen binding member comprising: a second antigen binding domain,e.g., a second antigen binding domain that binds a different antigenthan is bound by the antigen binding domain; and a second switch domain.

Also provided herein are RCARs wherein the antigen binding membercomprises bispecific activation and targeting capacity. In thisembodiment, the antigen binding member can comprise a plurality, e.g.,2, 3, 4, or 5 antigen binding domains, e.g., scFvs, wherein each antigenbinding domain binds to a target antigen, e.g. different antigens or thesame antigen, e.g., the same or different epitopes on the same antigen.In an embodiment, the plurality of antigen binding domains are intandem, and optionally, a linker or hinge region is disposed betweeneach of the antigen binding domains. Suitable linkers and hinge regionsare described herein.

An embodiment provides RCARs having a configuration that allowsswitching of proliferation. In this embodiment, the RCAR comprises: 1)an intracellular signaling member comprising: optionally, atransmembrane domain or membrane tethering domain; one or moreco-stimulatory signaling domain, e.g., selected from 4-1BB, CD28, CD27,ICOS, and OX40, and a switch domain; and 2) an antigen binding membercomprising: an antigen binding domain, a transmembrane domain, and aprimary intracellular signaling domain, e.g., a CD3zeta domain, whereinthe antigen binding member does not comprise a switch domain, or doesnot comprise a switch domain that dimerizes with a switch domain on theintracellular signaling member. In an embodiment, the antigen bindingmember does not comprise a co-stimulatory signaling domain. In anembodiment, the intracellular signaling member comprises a switch domainfrom a homodimerization switch. In an embodiment, the intracellularsignaling member comprises a first switch domain of a heterodimerizationswitch and the RCAR comprises a second intracellular signaling memberwhich comprises a second switch domain of the heterodimerization switch.In such embodiments, the second intracellular signaling member comprisesthe same intracellular signaling domains as the intracellular signalingmember. In an embodiment, the dimerization switch is intracellular. Inan embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described here, the first and secondswitch domains comprise a FKBP-FRB based switch as described herein.

Also provided herein are cells comprising an RCAR described herein. Anycell that is engineered to express a RCAR can be used as a RCARX cell.In an embodiment the RCARX cell is a T cell, and is referred to as aRCART cell. In an embodiment the RCARX cell is an NK cell, and isreferred to as a RCARN cell.

Also provided herein are nucleic acids and vectors comprising RCARencoding sequences. Sequence encoding various elements of an RCAR can bedisposed on the same nucleic acid molecule, e.g., the same plasmid orvector, e.g., viral vector, e.g., lentiviral vector. In an embodiment,(i) sequence encoding an antigen binding member and (ii) sequenceencoding an intracellular signaling member, can be present on the samenucleic acid, e.g., vector. Production of the corresponding proteins canbe achieved, e.g., by the use of separate promoters, or by the use of abicistronic transcription product (which can result in the production oftwo proteins by cleavage of a single translation product or by thetranslation of two separate protein products). In an embodiment, asequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, isdisposed between (i) and (ii). In an embodiment, a sequence encoding anIRES, e.g., an EMCV or EV71 IRES, is disposed between (i) and (ii). Inthese embodiments, (i) and (ii) are transcribed as a single RNA. In anembodiment, a first promoter is operably linked to (i) and a secondpromoter is operably linked to (ii), such that (i) and (ii) aretranscribed as separate mRNAs.

Alternatively, the sequence encoding various elements of an RCAR can bedisposed on the different nucleic acid molecules, e.g., differentplasmids or vectors, e.g., viral vector, e.g., lentiviral vector. E.g.,the (i) sequence encoding an antigen binding member can be present on afirst nucleic acid, e.g., a first vector, and the (ii) sequence encodingan intracellular signaling member can be present on the second nucleicacid, e.g., the second vector.

Dimerization Switches

Dimerization switches can be non-covalent or covalent. In a non-covalentdimerization switch, the dimerization molecule promotes a non-covalentinteraction between the switch domains. In a covalent dimerizationswitch, the dimerization molecule promotes a covalent interactionbetween the switch domains.

In an embodiment, the RCAR comprises a FKBP/FRAP, or FKBP/FRB,-baseddimerization switch. FKBP12 (FKBP, or FK506 binding protein) is anabundant cytoplasmic protein that serves as the initial intracellulartarget for the natural product immunosuppressive drug, rapamycin.Rapamycin binds to FKBP and to the large PI3K homolog FRAP (RAFT, mTOR).FRB is a 93 amino acid portion of FRAP, that is sufficient for bindingthe FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. &Schreiber, S. L. (1995) Identification of an 11-kDaFKBP12-rapamycin-binding domain within the 289-kDaFKBP12-rapamycin-associated protein and characterization of a criticalserine residue. Proc Natl Acad Sci USA 92: 4947-51.)

In embodiments, an FKBP/FRAP, e.g., an FKBP/FRB, based switch can use adimerization molecule, e.g., rapamycin or a rapamycin analog.

The amino acid sequence of FKBP is as follows:

(SEQ ID NO: 588) D V P D Y A S L G G P S S P K K K R K V S R G V QV E T I S P G D G R T F P K R G Q T C V V H Y T GM L E D G K K F D S S R D R N K P F K F M L G K QE V I R G W E E G V A Q M S V G Q R A K L T I S PD Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S Y

In embodiments, an FKBP switch domain can comprise a fragment of FKBPhaving the ability to bind with FRB, or a fragment or analog thereof, inthe presence of rapamycin or a rapalog, e.g., the underlined portion ofSEQ ID NO: 588, which is:

(SEQ ID NO: 589) V Q V E T I S P G D G R T F P K R G Q T C V V H YT G M L E D G K K F D S S R D R N K P F K F M L GK Q E V I R G W E E G V A Q M S V G Q R A K L T IS P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S

The amino acid sequence of FRB is as follows:

(SEQ ID NO: 590) ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMERGPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK

“FKBP/FRAP, e.g., an FKBP/FRB, based switch” as that term is usedherein, refers to a dimerization switch comprising: a first switchdomain, which comprises an FKBP fragment or analog thereof having theability to bind with FRB, or a fragment or analog thereof, in thepresence of rapamycin or a rapalog, e.g., RAD001, and has at least 70,75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by nomore than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from,the FKBP sequence of SEQ ID NO: 54 or 55; and a second switch domain,which comprises an FRB fragment or analog thereof having the ability tobind with FRB, or a fragment or analog thereof, in the presence ofrapamycin or a rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97,98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10,5, 4, 3, 2, or 1 amino acid residues from, the FRB sequence of SEQ IDNO: 56. In an embodiment, a RCAR described herein comprises one switchdomain comprises amino acid residues disclosed in SEQ ID NO: 588 (or SEQID NO: 589), and one switch domain comprises amino acid residuesdisclosed in SEQ ID NO: 590.

In embodiments, the FKBP/FRB dimerization switch comprises a modifiedFRB switch domain that exhibits altered, e.g., enhanced, complexformation between an FRB-based switch domain, e.g., the modified FRBswitch domain, a FKBP-based switch domain, and the dimerizationmolecule, e.g., rapamycin or a rapalogue, e.g., RAD001. In anembodiment, the modified FRB switch domain comprises one or moremutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected frommutations at amino acid position(s) L2031, E2032, 52035, R2036, F2039,G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type aminoacid is mutated to any other naturally-occurring amino acid. In anembodiment, a mutant FRB comprises a mutation at E2032, where E2032 ismutated to phenylalanine (E2032F), methionine (E2032M), arginine(E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g.,SEQ ID NO: 591, or leucine (E2032L), e.g., SEQ ID NO: 592. In anembodiment, a mutant FRB comprises a mutation at T2098, where T2098 ismutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO:593. In an embodiment, a mutant FRB comprises a mutation at E2032 and atT2098, where E2032 is mutated to any amino acid, and where T2098 ismutated to any amino acid, e.g., SEQ ID NO: 594. In an embodiment, amutant FRB comprises an E20321 and a T2098L mutation, e.g., SEQ ID NO:595. In an embodiment, a mutant FRB comprises an E2032L and a T2098Lmutation, e.g., SEQ ID NO: 596.

TABLE 14  Exemplary mutant FRB having increased affinityfor a dimerization molecule. SEQ FRB  ID mutant Amino Acid Sequence NO:E2032I ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHA 591 mutantMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG NVKDLTQAWDLYYHVFRRISKTS E2032LILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHA 592 mutantMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG NVKDLTQAWDLYYHVFRRISKTS T2098LILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHA 593 mutantMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG NVKDLLQAWDLYYHVFRRISKTS E2032,ILWHEMWHEGL X EASRLYFGERNVKGMFEVLEPLHA 594 T2098MMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG mutant NVKDL X QAWDLYYHVFRRISKTSE2032I, ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHA 595 T2098LMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG mutant NVKDLLQAWDLYYHVFRRISKTSE2032L, ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHA 596 T2098LMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG mutant NVKDLLQAWDLYYHVFRRISKTS

Other suitable dimerization switches include a GyrB-GyrB baseddimerization switch, a Gibberellin-based dimerization switch, atag/binder dimerization switch, and a halo-tag/snap-tag dimerizationswitch. Following the guidance provided herein, such switches andrelevant dimerization molecules will be apparent to one of ordinaryskill.

Dimerization Molecule

Association between the switch domains is promoted by the dimerizationmolecule. In the presence of dimerization molecule interaction orassociation between switch domains allows for signal transductionbetween a polypeptide associated with, e.g., fused to, a first switchdomain, and a polypeptide associated with, e.g., fused to, a secondswitch domain. In the presence of non-limiting levels of dimerizationmolecule signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 fold, e.g., as measured in asystem described herein.

Rapamycin and rapamycin analogs (sometimes referred to as rapalogues),e.g., RAD001, can be used as dimerization molecules in a FKBP/FRB-baseddimerization switch described herein. In an embodiment the dimerizationmolecule can be selected from rapamycin (sirolimus), RAD001(everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus),biolimus and AP21967. Additional rapamycin analogs suitable for use withFKBP/FRB-based dimerization switches are further described in thesection entitled “Combination Therapies”, or in the subsection entitled“Combination with a low dose mTOR inhibitor”.

Co-expression of CAR with a Chemokine Receptor

In embodiments, the CAR-expressing cell described herein furthercomprises a chemokine receptor molecule. Transgenic expression ofchemokine receptors CCR2b or CXCR2 in T cells enhances trafficking toCCL2- or CXCL1-secreting solid tumors including melanoma andneuroblastoma (Craddock et al., J Immunother. 2010 October; 33(8):780-8and Kershaw et al., Hum Gene Ther. 2002 Nov. 1; 13(16):1971-80). Thus,without wishing to be bound by theory, it is believed that chemokinereceptors expressed in CAR-expressing cells that recognize chemokinessecreted by tumors, e.g., solid tumors, can improve homing of theCAR-expressing cell to the tumor, facilitate the infiltration of theCAR-expressing cell to the tumor, and enhances antitumor efficacy of theCAR-expressing cell. The chemokine receptor molecule can comprise anaturally occurring or recombinant chemokine receptor or achemokine-binding fragment thereof. A chemokine receptor moleculesuitable for expression in a CAR-expressing cell described hereininclude a CXC chemokine receptor (e.g., CXCR1, CXCR2, CXCR3, CXCR4,CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (e.g., CCR1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), a CX3Cchemokine receptor (e.g., CX3CR1), a XC chemokine receptor (e.g., XCR1),or a chemokine-binding fragment thereof. In one embodiment, thechemokine receptor molecule to be expressed with a CAR described hereinis selected based on the chemokine(s) secreted by the tumor. In oneembodiment, the CAR-expressing cell described herein further comprises,e.g., expresses, a CCR2b receptor or a CXCR2 receptor. In an embodiment,the CAR described herein and the chemokine receptor molecule are on thesame vector or are on two different vectors. In embodiments where theCAR described herein and the chemokine receptor molecule are on the samevector, the CAR and the chemokine receptor molecule are each undercontrol of two different promoters or are under the control of the samepromoter.

RNA Transfection

Disclosed herein are methods for producing an in vitro transcribed RNACAR. The present invention also includes a CAR encoding RNA constructthat can be directly transfected into a cell. A method for generatingmRNA for use in transfection can involve in vitro transcription (IVT) ofa template with specially designed primers, followed by polyA addition,to produce a construct containing 3′ and 5′ untranslated sequence(“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), thenucleic acid to be expressed, and a polyA tail, typically 50-2000 basesin length (SEQ ID NO:35). RNA so produced can efficiently transfectdifferent kinds of cells. In one aspect, the template includes sequencesfor the CAR.

In one aspect the CD123 CAR is encoded by a messenger RNA (mRNA). In oneaspect the mRNA encoding the CD123 CAR is introduced into a T cell forproduction of a CART cell.

In one embodiment, the in vitro transcribed RNA CAR can be introduced toa cell as a form of transient transfection. The RNA is produced by invitro transcription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired temple for in vitrotranscription is a CAR of the present invention. For example, thetemplate for the RNA CAR comprises an extracellular region comprising asingle chain variable domain of an anti-tumor antibody; a hinge region,a transmembrane domain (e.g., a transmembrane domain of CD8a); and acytoplasmic region that includes an intracellular signaling domain,e.g., comprising the signaling domain of CD3-zeta and the signalingdomain of 4-1BB.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the nucleic acid can includesome or all of the 5′ and/or 3′ untranslated regions (UTRs). The nucleicacid can include exons and introns. In one embodiment, the DNA to beused for PCR is a human nucleic acid sequence. In another embodiment,the DNA to be used for PCR is a human nucleic acid sequence includingthe 5′ and 3′ UTRs. The DNA can alternatively be an artificial DNAsequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary,” as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5′ and 3′ UTRs.Primers useful for PCR can be generated by synthetic methods that arewell known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. Alternatively, UTR sequences thatare not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3′ UTR sequences candecrease the stability of mRNA. Therefore, 3′ UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5′ UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5′ UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3′or 5′ UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (SEQ ID NO: 31) (size can be 50-5000 T (SEQ ID NO: 32)), orafter PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Generally, the length of a poly(A)tail positively correlates with the stability of the transcribed RNA. Inone embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQID NO: 33).

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotides(SEQ ID NO: 34) results in about a two-fold increase in the translationefficiency of the RNA. Additionally, the attachment of differentchemical groups to the 3′ end can increase mRNA stability. Suchattachment can contain modified/artificial nucleotides, aptamers andother compounds. For example, ATP analogs can be incorporated into thepoly(A) tail using poly(A) polymerase. ATP analogs can further increasethe stability of the RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Non-viral Delivery Methods

In some aspects, non-viral methods can be used to deliver a nucleic acidencoding a CAR described herein into a cell or tissue or a subject.

In some embodiments, the non-viral method includes the use of atransposon (also called a transposable element). In some embodiments, atransposon is a piece of DNA that can insert itself at a location in agenome, for example, a piece of DNA that is capable of self-replicatingand inserting its copy into a genome, or a piece of DNA that can bespliced out of a longer nucleic acid and inserted into another place ina genome. For example, a transposon comprises a DNA sequence made up ofinverted repeats flanking genes for transposition.

Exemplary methods of nucleic acid delivery using a transposon include aSleeping Beauty transposon system (SBTS) and a piggyBac (PB) transposonsystem. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20;Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther.16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209;Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; andDing et al. Cell. 122.3(2005):473-83, all of which are incorporatedherein by reference.

The SBTS includes two components: 1) a transposon containing a transgeneand 2) a source of transposase enzyme. The transposase can transpose thetransposon from a carrier plasmid (or other donor DNA) to a target DNA,such as a host cell chromosome/genome. For example, the transposasebinds to the carrier plasmid/donor DNA, cuts the transposon (includingtransgene(s)) out of the plasmid, and inserts it into the genome of thehost cell. See, e.g., Aronovich et al. supra.

Exemplary transposons include a pT2-based transposon. See, e.g.,Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh etal. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporatedherein by reference. Exemplary transposases include a Tc1/mariner-typetransposase, e.g., the SB10 transposase or the SB11 transposase (ahyperactive transposase which can be expressed, e.g., from acytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.;and Grabundzija et al., all of which are incorporated herein byreference.

Use of the SBTS permits efficient integration and expression of atransgene, e.g., a nucleic acid encoding a CAR described herein.Provided herein are methods of generating a cell, e.g., T cell or NKcell, that stably expresses a CAR described herein, e.g., using atransposon system such as SBTS.

In accordance with methods described herein, in some embodiments, one ormore nucleic acids, e.g., plasmids, containing the SBTS components aredelivered to a cell (e.g., T or NK cell). For example, the nucleicacid(s) are delivered by standard methods of nucleic acid (e.g., plasmidDNA) delivery, e.g., methods described herein, e.g., electroporation,transfection, or lipofection. In some embodiments, the nucleic acidcontains a transposon comprising a transgene, e.g., a nucleic acidencoding a CAR described herein. In some embodiments, the nucleic acidcontains a transposon comprising a transgene (e.g., a nucleic acidencoding a CAR described herein) as well as a nucleic acid sequenceencoding a transposase enzyme. In other embodiments, a system with twonucleic acids is provided, e.g., a dual-plasmid system, e.g., where afirst plasmid contains a transposon comprising a transgene, and a secondplasmid contains a nucleic acid sequence encoding a transposase enzyme.For example, the first and the second nucleic acids are co-deliveredinto a host cell.

In some embodiments, cells, e.g., T or NK cells, are generated thatexpress a CAR described herein by using a combination of gene insertionusing the SBTS and genetic editing using a nuclease (e.g., Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALENs), the CRISPR/Cas system, or engineered meganucleasere-engineered homing endonucleases).

In some embodiments, use of a non-viral method of delivery permitsreprogramming of cells, e.g., T or NK cells, and direct infusion of thecells into a subject. Advantages of non-viral vectors include but arenot limited to the ease and relatively low cost of producing sufficientamounts required to meet a patient population, stability during storage,and lack of immunogenicity.

Nucleic Acid Constructs Encoding a CAR

The present invention also provides nucleic acid molecules encoding oneor more CAR constructs described herein. In one aspect, the nucleic acidmolecule is provided as a messenger RNA transcript. In one aspect, thenucleic acid molecule is provided as a DNA construct.

Accordingly, in one aspect, the invention pertains to an isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR),wherein the CAR comprises a CD123 binding domain (e.g., a humanized orhuman CD123 binding domain), a transmembrane domain, and anintracellular signaling domain comprising a stimulatory domain, e.g., acostimulatory signaling domain and/or a primary signaling domain, e.g.,zeta chain. In one embodiment, the CD123 binding domain is a CD123binding domain described herein, e.g., an CD123 binding domain whichcomprises a sequence selected from a group consisting of SEQ ID NO:157-160, 184-215, 478, 480, 483, 485, and 556-587, or a sequence with95-99% identity thereof. In one embodiment, the CD123 binding domaincomprises a human CD123 binding domain which comprises a sequenceselected from a group consisting of SEQ ID NO: 157-160, 478, 480, 483,and 485. In one embodiment, the CD123 binding domain comprises ahumanized CD123 binding domain which comprises a sequence selected froma group consisting of SEQ ID NO: 184-215, and 556-587. In oneembodiment, the transmembrane domain is transmembrane domain of aprotein, e.g., described herein, e.g., selected from the groupconsisting of the alpha, beta or zeta chain of the T-cell receptor,CD28, CD3 epsilon, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37,CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, thetransmembrane domain comprises a sequence of SEQ ID NO: 6, or a sequencewith 95-99% identity thereof. In one embodiment, the CD123 bindingdomain is connected to the transmembrane domain by a hinge region, e.g.,a hinge described herein. In one embodiment, the hinge region comprisesSEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5, or a sequencewith 95-99% identity thereof. In one embodiment, the isolated nucleicacid molecule further comprises a sequence encoding a costimulatorydomain. In one embodiment, the costimulatory domain is a functionalsignaling domain of a protein, e.g., described herein, e.g., selectedfrom the group consisting of a MHC class I molecule, a TNF receptorprotein, an Immunoglobulin-like protein, a cytokine receptor, anintegrin, a signaling lymphocytic activation molecule (SLAM protein), anactivating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2,CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB(CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specificallybinds with CD83.

In one embodiment, the costimulatory domain comprises a sequence of SEQID NO:7, or a sequence with 95-99% identity thereof. In one embodiment,the intracellular signaling domain comprises a functional signalingdomain of 4-1BB and a functional signaling domain of CD3 zeta. In oneembodiment, the intracellular signaling domain comprises the sequence ofSEQ ID NO: 7 or SEQ ID NO:8, or a sequence with 95-99% identity thereof,and the sequence of SEQ ID NO: 9 or SEQ ID NO:10, or a sequence with95-99% identity thereof, wherein the sequences comprising theintracellular signaling domain are expressed in the same frame and as asingle polypeptide chain.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence of SEQ IDNO: 1, a scFv domain having a sequence selected from the groupconsisting of SEQ ID NOS: 157-160, 184-215, 478, 480, 483, 485, and556-587, (or a sequence with 95-99% identity thereof), a hinge region ofSEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 (or a sequencewith 95-99% identity thereof), a transmembrane domain having a sequenceof SEQ ID NO: 6 (or a sequence with 95-99% identity thereof), a 4-1BBcostimulatory domain having a sequence of SEQ ID NO:7 or a CD27costimulatory domain having a sequence of SEQ ID NO:8 (or a sequencewith 95-99% identity thereof),) or a CD28 costimulatory domain having asequence of SEQ ID NO:43 (or a sequence with 95-99% identity thereof) ora ICOS costimulatory domain having a sequence of SEQ ID NO: 45 (or asequence with 95-99% identity thereof), and a CD3 zeta stimulatorydomain having a sequence of SEQ ID NO:9 or SEQ ID NO:10 (or a sequencewith 95-99% identity thereof).

In another aspect, the invention pertains to an isolated polypeptidemolecule encoded by the nucleic acid molecule. In one embodiment, theisolated polypeptide molecule comprises a sequence selected from thegroup consisting of SEQ ID NO:98-101 and 125-156, or a sequence with95-99% identity thereof.

In another aspect, the invention pertains to a nucleic acid moleculeencoding a chimeric antigen receptor (CAR) molecule that comprises aCD123 binding domain, a transmembrane domain, and an intracellularsignaling domain comprising a stimulatory domain, and wherein said CD123binding domain comprises a sequence selected from the group consistingof SEQ ID NO: 157-160, 184-215, 478, 480, 483, 485, and 556-587, or asequence with 95-99% identity thereof. In one embodiment, the CD123binding domain comprises a human CD123 binding domain comprising asequence selected from the group consisting of SEQ ID NO: 157-160, 478,480, 483, and 485, or a sequence with 95-99% identity thereof. In oneembodiment, the CD123 binding domain comprises a humanized CD123 bindingdomain comprising a sequence selected from the group consisting of SEQID NO: 184-215, and 556-587, or a sequence with 95-99% identity thereof.

In one embodiment, the encoded CAR molecule further comprises a sequenceencoding a costimulatory domain. In one embodiment, the costimulatorydomain is a functional signaling domain of a protein selected from thegroup consisting of a MHC class I molecule, a TNF receptor protein, anImmunoglobulin-like protein, a cytokine receptor, an integrin, asignaling lymphocytic activation molecule (SLAM protein), an activatingNK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.In one embodiment, the costimulatory domain comprises a sequence of SEQID NO:7.

In one embodiment, the transmembrane domain is a transmembrane domain ofa protein selected from the group consisting of the alpha, beta or zetachain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CD8,CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, aMHC class I molecule, a TNF receptor protein, an Immunoglobulin-likeprotein, a cytokine receptor, an integrin, a signaling lymphocyticactivation molecule (SLAM protein), an activating NK cell receptor,BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40,CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D,NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a,and a ligand that specifically binds with CD83.

In one embodiment, the transmembrane domain comprises a sequence of SEQID NO:6. In one embodiment, the intracellular signaling domain comprisesa functional signaling domain of 4-1BB and a functional signaling domainof zeta. In one embodiment, the intracellular signaling domain comprisesthe sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9, whereinthe sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain. In oneembodiment, the CD123 binding domain is connected to the transmembranedomain by a hinge region. In one embodiment, the hinge region comprisesSEQ ID NO:2. In one embodiment, the hinge region comprises SEQ ID NO:3or SEQ ID NO:4 or SEQ ID NO:5.

In another aspect, the invention pertains to an encoded CAR moleculecomprising a leader sequence of SEQ ID NO: 1, a scFv domain having asequence selected from the group consisting of SEQ ID NO: 157-160,184-215, 478, 480, 483, 485, 556-587, or a sequence with 95-99% identitythereof, a hinge region of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 orSEQ ID NO:5, a transmembrane domain having a sequence of SEQ ID NO: 6, a4-1BB costimulatory domain having a sequence of SEQ ID NO:7 or a CD27costimulatory domain having a sequence of SEQ ID NO:8 or a CD28costimulatory domain having a sequence of SEQ ID NO:43 or an ICOScostimulatory domain having a sequence of SEQ ID NO: 45, and a CD3 zetastimulatory domain having a sequence of SEQ ID NO:9 or SEQ ID NO:10. Inone embodiment, the encoded CAR molecule comprises a sequence selectedfrom a group consisting of SEQ ID NO: 98-101 and 125-156, or a sequencewith 95-99% identity thereof.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. A retroviral vector may also be, e.g., a gammaretroviralvector. A gammaretroviral vector may include, e.g., a promoter, apackaging signal (ψ), a primer binding site (PBS), one or more (e.g.,two) long terminal repeats (LTR), and a transgene of interest, e.g., agene encoding a CAR. A gammaretroviral vector may lack viral structuralgens such as gag, pol, and env. Exemplary gammaretroviral vectorsinclude Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV),and Myeloproliferative Sarcoma Virus (MPSV), and vectors derivedtherefrom. Other gammaretroviral vectors are described, e.g., in TobiasMaetzig et al., “Gammaretroviral Vectors: Biology, Technology andApplication” Viruses. 2011 June; 3(6): 677-713.

In another embodiment, the vector comprising the nucleic acid encodingthe desired CAR of the invention is an adenoviral vector (A5/35). Inanother embodiment, the expression of nucleic acids encoding CARs can beaccomplished using of transposons such as sleeping beauty, crisper,CAS9, and zinc finger nucleases. See below June et al. 2009 NatureReviews Immunology 9.10: 704-716, is incorporated herein by reference.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

An example of a promoter that is capable of expressing a CAR transgenein a mammalian T cell is the EF1a promoter. The native EF1a promoterdrives expression of the alpha subunit of the elongation factor-1complex, which is responsible for the enzymatic delivery of aminoacyltRNAs to the ribosome. The EF1a promoter has been extensively used inmammalian expression plasmids and has been shown to be effective indriving CAR expression from transgenes cloned into a lentiviral vector.See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In oneaspect, the EF1a promoter comprises the sequence provided as SEQ IDNO:11.

Another example of a promoter is the immediate early cytomegalovirus(CMV) promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the elongation factor-1apromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

Another example of a promoter is the phosphoglycerate kinase (PGK)promoter. In embodiments, a truncated PGK promoter (e.g., a PGK promoterwith one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotidedeletions when compared to the wild-type PGK promoter sequence) may bedesired. The nucleotide sequences of exemplary PGK promoters areprovided below.

WT PGK Promoter (SEQ ID NO: 597)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCGCGGCGACGCAAAGGGCCTTGGTGCGGGTCTCGTCGGCGCAGGGACGCGTTTGGGTCCCGACGGAACCTTTTCCGCGTTGGGGTTGGGGCACCATAAGCT Exemplary truncated PGK Promoters: PGK100: (SEQ ID NO: 598)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTG PGK200: (SEQ ID NO: 599)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACG PGK300: (SEQ ID NO: 600)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCG PGK400: (SEQ ID NO: 601)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCG

A vector may also include, e.g., a signal sequence to facilitatesecretion, a polyadenylation signal and transcription terminator (e.g.,from Bovine Growth Hormone (BGH) gene), an element allowing episomalreplication and replication in prokaryotes (e.g. SV40 origin and ColE1or others known in the art) and/or elements to allow selection (e.g.,ampicillin resistance gene and/or zeocin marker).

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In one embodiment, the vector can further comprise a nucleic acidencoding a second CAR. In one embodiment, the second CAR includes anantigen binding domain to a target expressed on acute myeloid leukemiacells, such as, e.g., CD33, CD34, CLL-1, FLT3, or folate receptor beta.In one embodiment, the vector comprises a nucleic acid sequence encodinga first CAR that targets a first antigen and includes an intracellularsignaling domain having a costimulatory signaling domain but not aprimary signaling domain, and a nucleic acid encoding a second CAR thattargets a second, different, antigen and includes an intracellularsignaling domain having a primary signaling domain but not acostimulatory signaling domain. In one embodiment, the vector comprisesa nucleic acid encoding a first CD123 CAR that includes a CD123 bindingdomain, a transmembrane domain and a costimulatory domain and a nucleicacid encoding a second CAR that targets an antigen other than CD123(e.g., an antigen expressed on AML cells, e.g., CD33, CD34, CLL-1, FLT3,or folate receptor beta) and includes an antigen binding domain, atransmembrane domain and a primary signaling domain. In anotherembodiment, the vector comprises a nucleic acid encoding a first CD123CAR that includes a CD123 binding domain, a transmembrane domain and aprimary signaling domain and a nucleic acid encoding a second CAR thattargets an antigen other than CD123 (e.g., an antigen expressed on AMLcells, e.g., CD33, CLL-1, CD34, FLT3, or folate receptor beta) andincludes an antigen binding domain to the antigen, a transmembranedomain and a costimulatory signaling domain.

In one embodiment, the vector comprises a nucleic acid encoding a CD123CAR described herein and a nucleic acid encoding an inhibitory CAR. Inone embodiment, the inhibitory CAR comprises an antigen binding domainthat binds an antigen found on normal cells but not cancer cells, e.g.,normal cells that also express CD123. In one embodiment, the inhibitoryCAR comprises the antigen binding domain, a transmembrane domain and anintracellular domain of an inhibitory molecule. For example, theintracellular domain of the inhibitory CAR can be an intracellulardomain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR,A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.

In embodiments, the vector may comprise two or more nucleic acidsequences encoding a CAR, e.g., a CD123 CAR described herein and asecond CAR, e.g., an inhibitory CAR or a CAR that specifically binds toan antigen other than CD123 (e.g., an antigen expressed on AML cells,e.g., CLL-1, CD33, CD34, FLT3, or folate receptor beta). In suchembodiments, the two or more nucleic acid sequences encoding the CAR areencoded by a single nucleic molecule in the same frame and as a singlepolypeptide chain. In this aspect, the two or more CARs, can, e.g., beseparated by one or more peptide cleavage sites. (e.g., an auto-cleavagesite or a substrate for an intracellular protease). Examples of peptidecleavage sites include the following, wherein the GSG residues areoptional:

T2A:  (SEQ ID NO: 602) (GSG) E G R G S L L T C G D V E E N P G P P2A: (SEQ ID NO: 603) (GSG) A T N F S L L K Q A G D V E E N P G PE2A: (SEQ ID NO: 604) (GSG) Q C T N Y A L L K L A G D V E S N P G P F2A: (SEQ ID NO: 605) (GSG) V K Q T L N F D L L K L A G D V E S N P G P

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

The present invention further provides a vector comprising a CARencoding nucleic acid molecule. In one aspect, a CAR vector can bedirectly transduced into a cell, e.g., an immune effector cell, e.g., aT cell or NK cell. In one aspect, the vector is a cloning or expressionvector, e.g., a vector including, but not limited to, one or moreplasmids (e.g., expression plasmids, cloning vectors, minicircles,minivectors, double minute chromosomes), retroviral and lentiviralvector constructs. In one aspect, the vector is capable of expressingthe CAR construct in mammalian immune effector cells, e.g., mammalian Tcells or mammalian NK cells. In one aspect, the mammalian T cell is ahuman T cell.

Sources of Cells

Prior to expansion and genetic modification, a source of cells (e.g.,immune effector cells, e.g., T cells or NK cells), is obtained from asubject. The term “subject” is intended to include living organisms inwhich an immune response can be elicited (e.g., mammals). Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors.

In certain aspects of the present invention, any number of immuneeffector cell (e.g., T cell or NK cell) lines available in the art, maybe used. In certain aspects of the present invention, T cells can beobtained from a unit of blood collected from a subject using any numberof techniques known to the skilled artisan, such as Ficoll™ separation.In one preferred aspect, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one aspect, the cells collected by apheresis may be washedto remove the plasma fraction and to place the cells in an appropriatebuffer or media for subsequent processing steps. In one aspect of theinvention, the cells are washed with phosphate buffered saline (PBS). Inan alternative aspect, the wash solution lacks calcium and may lackmagnesium or may lack many if not all divalent cations. Initialactivation steps in the absence of calcium can lead to magnifiedactivation. As those of ordinary skill in the art would readilyappreciate a washing step may be accomplished by methods known to thosein the art, such as by using a semi-automated “flow-through” centrifuge(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions.After washing, the cells may be resuspended in a variety ofbiocompatible buffers, such as, for example, Ca-free, Mg-free PBS,PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may beremoved and the cells directly resuspended in culture media.

It is recognized that the methods of the application can utilize culturemedia conditions comprising 5% or less, for example 2%, human AB serum,and employ known culture media conditions and compositions, for examplethose described in Smith et al., “Ex vivo expansion of human T cells foradoptive immunotherapy using the novel Xeno-free CTS Immune Cell SerumReplacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038/cti.2014.31.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation. A specific subpopulation of T cells, such as CD3+, CD28+,CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated bypositive or negative selection techniques. For example, in one aspect, Tcells are isolated by incubation with anti-CD3/anti-CD28 (e.g.,3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a timeperiod sufficient for positive selection of the desired T cells. In oneaspect, the time period is about 30 minutes. In a further aspect, thetime period ranges from 30 minutes to 36 hours or longer and all integervalues there between. In a further aspect, the time period is at least1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the timeperiod is 10 to 24 hours. In one aspect, the incubation time period is24 hours. Longer incubation times may be used to isolate T cells in anysituation where there are few T cells as compared to other cell types,such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissueor from immunocompromised individuals. Further, use of longer incubationtimes can increase the efficiency of capture of CD8+ T cells. Thus, bysimply shortening or lengthening the time T cells are allowed to bind tothe CD3/CD28 beads and/or by increasing or decreasing the ratio of beadsto T cells (as described further herein), subpopulations of T cells canbe preferentially selected for or against at culture initiation or atother time points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainaspects, it may be desirable to perform the selection procedure and usethe “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain aspects, it may be desirable to enrich foror positively select for regulatory T cells which typically expressCD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certainaspects, T regulatory cells are depleted by anti-C25 conjugated beads orother similar method of selection.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. Preferably, thepopulation of T regulatory depleted cells contains less than 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removedfrom the population using an anti-CD25 antibody, or fragment thereof, ora CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody,or fragment thereof, or CD25-binding ligand is conjugated to asubstrate, e.g., a bead, or is otherwise coated on a substrate, e.g., abead. In one embodiment, the anti-CD25 antibody, or fragment thereof, isconjugated to a substrate as described herein.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using CD25 depletion reagent from Miltenyi™.In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7cells to 20 uL, or 1e7 cells to15 uL, or 1e7 cells to 10 uL, or 1e7cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In oneembodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greaterthan 500 million cells/ml is used. In a further aspect, a concentrationof cells of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to bedepleted includes about 6×10⁹ CD25+ T cells. In other aspects, thepopulation of immune effector cells to be depleted include about 1×10⁹to 1× 10¹⁰ CD25+ T cell, and any integer value in between. In oneembodiment, the resulting population T regulatory depleted cells has2×10⁹ T regulatory cells, e.g., CD25+ cells, or less (e.g., 1×10⁹,5×10⁸, 1×10⁸, 5×10⁷, 1×10⁷, or less CD25+ cells).

In one embodiment, the T regulatory cells, e.g., CD25+ cells, areremoved from the population using the CliniMAC system with a depletiontubing set, such as, e.g., tubing 162-01. In one embodiment, theCliniMAC system is run on a depletion setting such as, e.g.,DEPLETION2.1.

Without wishing to be bound by a particular theory, decreasing the levelof negative regulators of immune cells (e.g., decreasing the number ofunwanted immune cells, e.g., T_(REG) cells), in a subject prior toapheresis or during manufacturing of a CAR-expressing cell product canreduce the risk of subject relapse. For example, methods of depletingT_(REG) cells are known in the art. Methods of decreasing T_(REG) cellsinclude, but are not limited to, cyclophosphamide, anti-GITR antibody(an anti-GITR antibody described herein), CD25-depletion, andcombinations thereof.

In some embodiments, the manufacturing methods comprise reducing thenumber of (e.g., depleting) T_(REG) cells prior to manufacturing of theCAR-expressing cell. For example, manufacturing methods comprisecontacting the sample, e.g., the apheresis sample, with an anti-GITRantibody and/or an anti-CD25 antibody (or fragment thereof, or aCD25-binding ligand), e.g., to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell)product.

In an embodiment, a subject is pre-treated with one or more therapiesthat reduce T_(REG) cells prior to collection of cells forCAR-expressing cell product manufacturing, thereby reducing the risk ofsubject relapse to CAR-expressing cell treatment. In an embodiment,methods of decreasing T_(REG) cells include, but are not limited to,administration to the subject of one or more of cyclophosphamide,anti-GITR antibody, CD25-depletion, or a combination thereof.Administration of one or more of cyclophosphamide, anti-GITR antibody,CD25-depletion, or a combination thereof, can occur before, during orafter an infusion of the CAR-expressing cell product.

In an embodiment, a subject is pre-treated with cyclophosphamide priorto collection of cells for CAR-expressing cell product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment. In an embodiment, a subject is pre-treated with an anti-GITRantibody prior to collection of cells for CAR-expressing cell productmanufacturing, thereby reducing the risk of subject relapse toCAR-expressing cell treatment.

In one embodiment, the population of cells to be removed are neither theregulatory T cells or tumor cells, but cells that otherwise negativelyaffect the expansion and/or function of CART cells, e.g. cellsexpressing CD14, CD11b, CD33, CD15, or other markers expressed bypotentially immune suppressive cells. In one embodiment, such cells areenvisioned to be removed concurrently with regulatory T cells and/ortumor cells, or following said depletion, or in another order.

The methods described herein can include more than one selection step,e.g., more than one depletion step. Enrichment of a T cell population bynegative selection can be accomplished, e.g., with a combination ofantibodies directed to surface markers unique to the negatively selectedcells. One method is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail can include antibodies toCD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein can further include removing cells from thepopulation which express a tumor antigen, e.g., a tumor antigen thatdoes not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 orCD11b, to thereby provide a population of T regulatory depleted, e.g.,CD25+ depleted, and tumor antigen depleted cells that are suitable forexpression of a CAR, e.g., a CAR described herein. In one embodiment,tumor antigen expressing cells are removed simultaneously with the Tregulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, orfragment thereof, and an anti-tumor antigen antibody, or fragmentthereof, can be attached to the same substrate, e.g., bead, which can beused to remove the cells or an anti-CD25 antibody, or fragment thereof,or the anti-tumor antigen antibody, or fragment thereof, can be attachedto separate beads, a mixture of which can be used to remove the cells.In other embodiments, the removal of T regulatory cells, e.g., CD25+cells, and the removal of the tumor antigen expressing cells issequential, and can occur, e.g., in either order.

Also provided are methods that include removing cells from thepopulation which express a check point inhibitor, e.g., a check pointinhibitor described herein, e.g., one or more of PD1+ cells, LAG3+cells, and TIM3+ cells, to thereby provide a population of T regulatorydepleted, e.g., CD25+ depleted cells, and check point inhibitor depletedcells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLAand LAIR1. In one embodiment, check point inhibitor expressing cells areremoved simultaneously with the T regulatory, e.g., CD25+ cells. Forexample, an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be attached to thesame bead which can be used to remove the cells, or an anti-CD25antibody, or fragment thereof, and the anti-check point inhibitorantibody, or fragment there, can be attached to separate beads, amixture of which can be used to remove the cells. In other embodiments,the removal of T regulatory cells, e.g., CD25+ cells, and the removal ofthe check point inhibitor expressing cells is sequential, and can occur,e.g., in either order.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10,IL-13, granzyme B, and perforin, or other appropriate molecules, e.g.,other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 2 billion cells/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In a further aspect, greater than 100 millioncells/ml is used. In a further aspect, a concentration of cells of 10,15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet oneaspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 millioncells/ml is used. In further aspects, concentrations of 125 or 150million cells/ml can be used. Using high concentrations can result inincreased cell yield, cell activation, and cell expansion. Further, useof high cell concentrations allows more efficient capture of cells thatmay weakly express target antigens of interest, such as CD28-negative Tcells, or from samples where there are many tumor cells present (e.g.,leukemic blood, tumor tissue, etc.). Such populations of cells may havetherapeutic value and would be desirable to obtain. For example, usinghigh concentration of cells allows more efficient selection of CD8+ Tcells that normally have weaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10e6/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as immune effector cells, e.g.,T cells or NK cells, isolated and frozen for later use in T cell therapyfor any number of diseases or conditions that would benefit from celltherapy, e.g., T cell therapy, such as those described herein. In oneaspect a blood sample or an apheresis is taken from a generally healthysubject. In certain aspects, a blood sample or an apheresis is takenfrom a generally healthy subject who is at risk of developing a disease,but who has not yet developed a disease, and the cells of interest areisolated and frozen for later use. In certain aspects, the immuneeffector cells, e.g., T cells or NK cells, may be expanded, frozen, andused at a later time. In certain aspects, samples are collected from apatient shortly after diagnosis of a particular disease as describedherein but prior to any treatments. In a further aspect, the cells areisolated from a blood sample or an apheresis from a subject prior to anynumber of relevant treatment modalities, including but not limited totreatment with agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, including Tcells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T cells, B cells, dendriticcells, and other cells of the immune system.

In one embodiment, the immune effector cells expressing a CAR molecule,e.g., a CAR molecule described herein, are obtained from a subject thathas received a low, immune enhancing dose of an mTOR inhibitor. In anembodiment, the population of immune effector cells, e.g., T cells, tobe engineered to express a CAR, are harvested after a sufficient time,or after sufficient dosing of the low, immune enhancing, dose of an mTORinhibitor, such that the level of PD1 negative immune effector cells,e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g.,T cells/PD1 positive immune effector cells, e.g., T cells, in thesubject or harvested from the subject has been, at least transiently,increased.

In other embodiments, population of immune effector cells, e.g., Tcells, which have, or will be engineered to express a CAR, can betreated ex vivo by contact with an amount of an mTOR inhibitor thatincreases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

In one embodiment, a T cell population is diaglycerol kinase(DGK)-deficient. DGK-deficient cells include cells that do not expressDGK RNA or protein, or have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, e.g.,administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, toreduce or prevent DGK expression. Alternatively, DGK-deficient cells canbe generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA orprotein, or have reduced or inhibited Ikaros activity, Ikaros-deficientcells can be generated by genetic approaches, e.g., administeringRNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or preventIkaros expression. Alternatively, Ikaros-deficient cells can begenerated by treatment with Ikaros inhibitors, e.g., lenalidomide.

In embodiments, a T cell population is DGK-deficient andIkaros-deficient, e.g., does not express DGK and Ikaros, or has reducedor inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficientcells can be generated by any of the methods described herein.

In an embodiment, the NK cells are obtained from the subject. In anotherembodiment, the NK cells are an NK cell line, e.g., NK-92 cell line(Conkwest).

Allogeneic CAR Immune Effector Cells

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell. For example,the cell can be an allogeneic T cell, e.g., an allogeneic T cell lackingexpression of a functional T cell receptor (TCR) and/or human leukocyteantigen (HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional TCR can be, e.g., engineered such that itdoes not express any functional TCR on its surface, engineered such thatit does not express one or more subunits that comprise a functional TCR(e.g., engineered such that it does not express (or exhibits reducedexpression) of TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon,and/or TCR zeta) or engineered such that it produces very littlefunctional TCR on its surface. Alternatively, the T cell can express asubstantially impaired TCR, e.g., by expression of mutated or truncatedforms of one or more of the subunits of the TCR. The term “substantiallyimpaired TCR” means that this TCR will not elicit an adverse immunereaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated. In some aspects,downregulation of HLA may be accomplished by reducing or eliminatingexpression of beta-2 microglobulin (B2M).

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II.

Modified T cells that lack expression of a functional TCR and/or HLA canbe obtained by any suitable means, including a knock out or knock downof one or more subunit of TCR or HLA. For example, the T cell caninclude a knock down of TCR and/or HLA using siRNA, shRNA, clusteredregularly interspaced short palindromic repeats (CRISPR)transcription-activator like effector nuclease (TALEN), or zinc fingerendonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpresses or expresses at low levels an inhibitory molecule, e.g. by anymethod described herein. For example, the cell can be a cell that doesnot express or expresses at low levels an inhibitory molecule, e.g.,that can decrease the ability of a CAR-expressing cell to mount animmune effector response. Examples of inhibitory molecules include PD1,PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR beta. Inhibition of aninhibitory molecule, e.g., by inhibition at the DNA, RNA or proteinlevel, can optimize a CAR-expressing cell performance. In embodiments,an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., adsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used.

siRNA and shRNA to Inhibit TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR beta), in a cell.

Expression of siRNA and shRNAs in T cells can be achieved using anyconventional expression system, e.g., such as a lentiviral expressionsystem.

Exemplary shRNAs that downregulate expression of one or more componentsof the TCR are described, e.g., in US Publication No.: 2012/0321667.Exemplary siRNA and shRNA that downregulate expression of HLA class Iand/or HLA class II genes are described, e.g., in U.S. publication No.:US 2007/0036773.

CRISPR to Inhibit TCR or HLA

“CRISPR” or “CRISPR to TCR and/or HLA” or “CRISPR to inhibit TCR and/orHLA” as used herein refers to a set of clustered regularly interspacedshort palindromic repeats, or a system comprising such a set of repeats.“Cas”, as used herein, refers to a CRISPR-associated protein. A“CRISPR/Cas” system refers to a system derived from CRISPR and Cas whichcan be used to silence or mutate a TCR and/or HLA gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta).

Naturally-occurring CRISPR/Cas systems are found in approximately 40% ofsequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al.(2007) BMC Bioinformatics 8: 172. This system is a type of prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity.Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008)Science 322: 1843-1845.

The CRISPR/Cas system has been modified for use in gene editing(silencing, enhancing or changing specific genes) in eukaryotes such asmice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This isaccomplished by introducing into the eukaryotic cell a plasmidcontaining a specifically designed CRISPR and one or more appropriateCas.

The CRISPR sequence, sometimes called a CRISPR locus, comprisesalternating repeats and spacers. In a naturally-occurring CRISPR, thespacers usually comprise sequences foreign to the bacterium such as aplasmid or phage sequence; in the TCR and/or HLA CRISPR/Cas system, thespacers are derived from the TCR or HLA gene sequence.

RNA from the CRISPR locus is constitutively expressed and processed byCas proteins into small RNAs. These comprise a spacer flanked by arepeat sequence. The RNAs guide other Cas proteins to silence exogenousgenetic elements at the RNA or DNA level. Horvath et al. (2010) Science327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacersthus serve as templates for RNA molecules, analogously to siRNAs.Pennisi (2013) Science 341: 833-836.

As these naturally occur in many different types of bacteria, the exactarrangements of the CRISPR and structure, function and number of Casgenes and their product differ somewhat from species to species. Haft etal. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol.8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al.(2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. Forexample, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. Brouns et al. (2008) Science321: 960-964. In other prokaryotes, Cas6 processes the CRISPRtranscript. The CRISPR-based phage inactivation in E. coli requiresCascade and Cas3, but not Cast or Cas2. The Cmr (Cas RAMP module)proteins in Pyrococcus furiosus and other prokaryotes form a functionalcomplex with small CRISPR RNAs that recognizes and cleaves complementarytarget RNAs. A simpler CRISPR system relies on the protein Cas9, whichis a nuclease with two active cutting sites, one for each strand of thedouble helix. Combining Cas9 and modified CRISPR locus RNA can be usedin a system for gene editing. Pennisi (2013) Science 341: 833-836.

The CRISPR/Cas system can thus be used to edit a TCR and/or HLA gene(adding or deleting a basepair), or introducing a premature stop whichthus decreases expression of a TCR and/or HLA. The CRISPR/Cas system canalternatively be used like RNA interference, turning off TCR and/or HLAgene in a reversible fashion. In a mammalian cell, for example, the RNAcan guide the Cas protein to a TCR and/or HLA promoter, stericallyblocking RNA polymerases.

Artificial CRISPR/Cas systems can be generated which inhibit TCR and/orHLA, using technology known in the art, e.g., that described in U.S.Publication No. 20140068797 and Cong (2013) Science 339: 819-823. Otherartificial CRISPR/Cas systems that are known in the art may also begenerated which inhibit TCR and/or HLA, e.g., that described in Tsai(2014) Nature Biotechnol., 32:6 569-576, U.S. Pat. Nos. 8,871,445;8,865,406; 8,795,965; 8,771,945; and 8,697,359.

TALEN to Inhibit TCR and/or HLA

“TALEN” or “TALEN to HLA and/or TCR” or “TALEN to inhibit HLA and/orTCR” refers to a transcription activator-like effector nuclease, anartificial nuclease which can be used to edit the HLA and/or TCR gene,and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta).

TALENs are produced artificially by fusing a TAL effector DNA bindingdomain to a DNA cleavage domain. Transcription activator-like effects(TALEs) can be engineered to bind any desired DNA sequence, including aportion of the HLA or TCR gene. By combining an engineered TALE with aDNA cleavage domain, a restriction enzyme can be produced which isspecific to any desired DNA sequence, including a HLA or TCR sequence.These can then be introduced into a cell, wherein they can be used forgenome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al.(2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.

TALEs are proteins secreted by Xanthomonas bacteria. The DNA bindingdomain contains a repeated, highly conserved 33-34 amino acid sequence,with the exception of the 12th and 13th amino acids. These two positionsare highly variable, showing a strong correlation with specificnucleotide recognition. They can thus be engineered to bind to a desiredDNA sequence.

To produce a TALEN, a TALE protein is fused to a nuclease (N), which isa wild-type or mutated FokI endonuclease. Several mutations to FokI havebeen made for its use in TALENs; these, for example, improve cleavagespecificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82;Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011)Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyonet al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) NatureBiotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96.

The FokI domain functions as a dimer, requiring two constructs withunique DNA binding domains for sites in the target genome with properorientation and spacing. Both the number of amino acid residues betweenthe TALE DNA binding domain and the FokI cleavage domain and the numberof bases between the two individual TALEN binding sites appear to beimportant parameters for achieving high levels of activity. Miller etal. (2011) Nature Biotech. 29: 143-8.

A HLA or TCR TALEN can be used inside a cell to produce adouble-stranded break (DSB). A mutation can be introduced at the breaksite if the repair mechanisms improperly repair the break vianon-homologous end joining. For example, improper repair may introduce aframe shift mutation. Alternatively, foreign DNA can be introduced intothe cell along with the TALEN; depending on the sequences of the foreignDNA and chromosomal sequence, this process can be used to correct adefect in the HLA or TCR gene or introduce such a defect into a wt HLAor TCR gene, thus decreasing expression of HLA or TCR.

TALENs specific to sequences in HLA or TCR can be constructed using anymethod known in the art, including various schemes using modularcomponents. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler etal. (2011) PLoS ONE 6: e19509.

Zinc Finger Nuclease to Inhibit HLA and/or TCR

“ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and/or TCR” or “ZFN toinhibit HLA and/or TCR” refer to a zinc finger nuclease, an artificialnuclease which can be used to edit the HLA and/or TCR gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta).

Like a TALEN, a ZFN comprises a FokI nuclease domain (or derivativethereof) fused to a DNA-binding domain. In the case of a ZFN, theDNA-binding domain comprises one or more zinc fingers. Carroll et al.(2011) Genetics Society of America 188: 773-782; and Kim et al. (1996)Proc. Natl. Acad. Sci. USA 93: 1156-1160.

A zinc finger is a small protein structural motif stabilized by one ormore zinc ions. A zinc finger can comprise, for example, Cys2His2, andcan recognize an approximately 3-bp sequence. Various zinc fingers ofknown specificity can be combined to produce multi-finger polypeptideswhich recognize about 6, 9, 12, 15 or 18-bp sequences. Various selectionand modular assembly techniques are available to generate zinc fingers(and combinations thereof) recognizing specific sequences, includingphage display, yeast one-hybrid systems, bacterial one-hybrid andtwo-hybrid systems, and mammalian cells.

Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNsare required to target non-palindromic DNA sites. The two individualZFNs must bind opposite strands of the DNA with their nucleases properlyspaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10570-5.

Also like a TALEN, a ZFN can create a double-stranded break in the DNA,which can create a frame-shift mutation if improperly repaired, leadingto a decrease in the expression and amount of HLA and/or TCR in a cell.ZFNs can also be used with homologous recombination to mutate in the HLAor TCR gene.

ZFNs specific to sequences in HLA AND/OR TCR can be constructed usingany method known in the art. Cathomen et al. (2008) Mol. Ther. 16:1200-7; Guo et al. (2010) J. Mol. Biol. 400: 96; U.S. Patent Publication2011/0158957; and U.S. Patent Publication 2012/0060230.

Telomerase Expression

While not wishing to be bound by any particular theory, in someembodiments, a therapeutic T cell has short term persistence in apatient, due to shortened telomeres in the T cell; accordingly,transfection with a telomerase gene can lengthen the telomeres of the Tcell and improve persistence of the T cell in the patient. See CarlJune, “Adoptive T cell therapy for cancer in the clinic”, Journal ofClinical Investigation, 117:1466-1476 (2007). Thus, in an embodiment, animmune effector cell, e.g., a T cell, ectopically expresses a telomerasesubunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g.,hTERT. In some aspects, this disclosure provides a method of producing aCAR-expressing cell, comprising contacting a cell with a nucleic acidencoding a telomerase subunit, e.g., the catalytic subunit oftelomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with thenucleic acid before, simultaneous with, or after being contacted with aconstruct encoding a CAR.

In one aspect, the disclosure features a method of making a populationof immune effector cells (e.g., T cells, NK cells). In an embodiment,the method comprises: providing a population of immune effector cells(e.g., T cells or NK cells), contacting the population of immuneeffector cells with a nucleic acid encoding a CAR; and contacting thepopulation of immune effector cells with a nucleic acid encoding atelomerase subunit, e.g., hTERT, under conditions that allow for CAR andtelomerase expression.

In an embodiment, the nucleic acid encoding the telomerase subunit isDNA. In an embodiment, the nucleic acid encoding the telomerase subunitcomprises a promoter capable of driving expression of the telomerasesubunit.

In an embodiment, hTERT has the amino acid sequence of GenBank ProteinID AAC51724.1 (Meyerson et al., “hEST2, the Putative Human TelomeraseCatalytic Subunit Gene, Is Up-Regulated in Tumor Cells and duringImmortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795)as follows:

(SEQ ID NO: 606) MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD 

In an embodiment, the hTERT has a sequence at least 80%, 85%, 90%, 95%,96^, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 606. In anembodiment, the hTERT has a sequence of SEQ ID NO: 606. In anembodiment, the hTERT comprises a deletion (e.g., of no more than 5, 10,15, 20, or 30 amino acids) at the N-terminus, the C-terminus, or both.In an embodiment, the hTERT comprises a transgenic amino acid sequence(e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at theN-terminus, the C-terminus, or both.

In an embodiment, the hTERT is encoded by the nucleic acid sequence ofGenBank Accession No. AF018167 (Meyerson et al., “hEST2, the PutativeHuman Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cellsand during Immortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages785-795):

(SEQ ID NO: 607)   1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc  61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc 121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg 241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg 361 cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct 421 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc 481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg 661 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga 721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg 781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga 841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 901 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc 961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg1141 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt1741 atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga1801 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag1981 ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc2221 aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa2701 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga2761 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg2881 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc3541 ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc3721 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa4021 aaaaaaa 

In an embodiment, the hTERT is encoded by a nucleic acid having asequence at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 607. In an embodiment, the hTERT is encodedby a nucleic acid of SEQ ID NO: 607.

Activation and Expansion of Cells

Cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the T cells of the invention may be expanded by contact witha surface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody can be used. Examples of an anti-CD28 antibodyinclude 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France) can be used ascan other methods commonly known in the art (Berg et al., TransplantProc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63,1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain aspects of theinvention, the ratio of anti CD28 antibody to anti CD3 antibody bound tothe beads is greater than 2:1. In one particular aspect, a 1:100CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a further aspect,a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect,a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In onepreferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beadsis used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound tothe beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle: cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects of the present invention, the cells, such as T cells,are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative aspect, prior to culture, the agent-coated beads and cellsare not separated but are cultured together. In a further aspect, thebeads and cells are first concentrated by application of a force, suchas a magnetic force, resulting in increased ligation of cell surfacemarkers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example, 10⁴to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect,greater than 100 million cells/ml is used. In a further aspect, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet one aspect, a concentration of cells from 75,80, 85, 90, 95, or 100 million cells/ml is used. In further aspects,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainaspects. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment, cells transduced with a nucleic acid encoding a CAR,e.g., a CAR described herein, are expanded, e.g., by a method describedherein. In one embodiment, the cells are expanded in culture for aperiod of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18,21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days). In one embodiment, the cells are expanded for a periodof 4 to 9 days. In one embodiment, the cells are expanded for a periodof 8 days or less, e.g., 7, 6 or 5 days. In one embodiment, the cells,e.g., a CD123 CAR cell described herein, are expanded in culture for 5days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions.Potency can be defined, e.g., by various T cell functions, e.g.proliferation, target cell killing, cytokine production, activation,migration, or combinations thereof. In one embodiment, the cells, e.g.,a CD123 CAR cell described herein, expanded for 5 days show at least aone, two, three or four fold increase in cells doublings upon antigenstimulation as compared to the same cells expanded in culture for 9 daysunder the same culture conditions. In one embodiment, the cells, e.g.,the cells expressing a CD123 CAR described herein, are expanded inculture for 5 days, and the resulting cells exhibit higherproinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels,as compared to the same cells expanded in culture for 9 days under thesame culture conditions. In one embodiment, the cells, e.g., a CD123 CARcell described herein, expanded for 5 days show at least a one, two,three, four, five, ten fold or more increase in pg/ml of proinflammatorycytokine production, e.g., IFN-γ and/or GM-CSF levels, as compared tothe same cells expanded in culture for 9 days under the same cultureconditions.

In one aspect of the present invention, the mixture may be cultured forseveral hours (about 3 hours) to about 14 days or any hourly integervalue in between. In one aspect, the mixture may be cultured for 21days. In one aspect of the invention the beads and the T cells arecultured together for about eight days. In one aspect, the beads and Tcells are cultured together for 2-3 days. Several cycles of stimulationmay also be desired such that culture time of T cells can be 60 days ormore. Conditions appropriate for T cell culture include an appropriatemedia (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that includes one or more interleukin thatresult in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold,350-fold) increase in cells over a 14 day expansion period, e.g., asmeasured by a method described herein such as flow cytometry. In oneembodiment, the cells are expanded in the presence of IL-15 and/or IL-7(e.g., IL-15 and IL-7).

In embodiments, methods described herein, e.g., CAR-expressing cellmanufacturing methods, comprise removing T regulatory cells, e.g., CD25+T cells, from a cell population, e.g., using an anti-CD25 antibody, orfragment thereof, or a CD25-binding ligand, IL-2. Methods of removing Tregulatory cells, e.g., CD25+ T cells, from a cell population aredescribed herein. In embodiments, the methods, e.g., manufacturingmethods, further comprise contacting a cell population (e.g., a cellpopulation in which T regulatory cells, such as CD25+ T cells, have beendepleted; or a cell population that has previously contacted ananti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15and/or IL-7. For example, the cell population (e.g., that has previouslycontacted an anti-CD25 antibody, fragment thereof, or CD25-bindingligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments a CAR-expressing cell described herein is contactedwith a composition comprising a interleukin-15 (IL-15) polypeptide, ainterleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination ofboth a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15,during the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising a IL-15 polypeptide during the manufacturing ofthe CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressingcell described herein is contacted with a composition comprising acombination of both a IL-15 polypeptide and a IL-15 Ra polypeptideduring the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising hetIL-15 during the manufacturing of theCAR-expressing cell, e.g., ex vivo.

In one embodiment the CAR-expressing cell described herein is contactedwith a composition comprising hetIL-15 during ex vivo expansion. In anembodiment, the CAR-expressing cell described herein is contacted with acomposition comprising an IL-15 polypeptide during ex vivo expansion. Inan embodiment, the CAR-expressing cell described herein is contactedwith a composition comprising both an IL-15 polypeptide and an IL-15Rapolypeptide during ex vivo expansion. In one embodiment the contactingresults in the survival and proliferation of a lymphocyte subpopulation,e.g., CD8+ T cells.

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Once a CD123 CAR is constructed, various assays can be used to evaluatethe activity of the molecule, such as but not limited to, the ability toexpand T cells following antigen stimulation, sustain T cell expansionin the absence of re-stimulation, and anti-cancer activities inappropriate in vitro and animal models. Assays to evaluate the effectsof a CD123 CAR are described in further detail below.

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1mixture of CD4⁺ and CD8⁺ T cells) expressing the CARs are expanded invitro for more than 10 days followed by lysis and SDS-PAGE underreducing conditions. CARs containing the full length TCR-ζ cytoplasmicdomain and the endogenous TCR-ζ chain are detected by western blottingusing an antibody to the TCR-ζ chain. The same T cell subsets are usedfor SDS-PAGE analysis under non-reducing conditions to permit evaluationof covalent dimer formation.

In vitro expansion of CAR⁺ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD3/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1α,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated with either CD19⁺ K562 cells (K562-CD19),wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and4-1BBL in the presence of antiCD3 and anti-CD28 antibody (K562-BBL-3/28)following washing. Exogenous IL-2 is added to the cultures every otherday at 100 IU/ml. GFP⁺ T cells are enumerated by flow cytometry usingbead-based counting. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Similar assays can be performed using anti-CD123 Tcells (see, e.g. Gill et al Blood 2014; 123:2343) or with anti-CD123 CART cells.

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter, a NexcelomCellometer Vision or Millipore Scepter, following stimulation withαCD3/αCD28 coated magnetic beads on day 0, and transduction with theindicated CAR on day 1.

Animal models can also be used to measure a CART activity. For example,xenograft model using human CD19-specific CAR⁺ T cells to treat aprimary human pre-B ALL in immunodeficient mice can be used. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly,after establishment of ALL, mice are randomized as to treatment groups.Different numbers of αCD19-ζ and αCD19-BB-ζ engineered T cells arecoinjected at a 1:1 ratio into NOD-SCID-γ^(−/−) mice bearing B-ALL. Thenumber of copies of αCD19-ζ and αCD19-BB-ζ vector in spleen DNA frommice is evaluated at various times following T cell injection. Animalsare assessed for leukemia at weekly intervals. Peripheral blood CD19⁺B-ALL blast cell counts are measured in mice that are injected withαCD19-ζ CAR⁺ T cells or mock-transduced T cells. Survival curves for thegroups are compared using the log-rank test. In addition, absoluteperipheral blood CD4⁺ and CD8⁺ T cell counts 4 weeks following T cellinjection in NOD-SCID-γ^(−/−) mice can also be analyzed. Mice areinjected with leukemic cells and 3 weeks later are injected with T cellsengineered to express CAR by a bicistronic lentiviral vector thatencodes the CAR linked to eGFP. T cells are normalized to 45-50% inputGFP⁺ T cells by mixing with mock-transduced cells prior to injection,and confirmed by flow cytometry. Animals are assessed for leukemia at1-week intervals. Survival curves for the CAR⁺ T cell groups arecompared using the log-rank test. Similar experiments can be done withCD123 CARTS.

Dose dependent CAR treatment response can be evaluated. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example,peripheral blood is obtained 35-70 days after establishing leukemia inmice injected on day 21 with CAR T cells, an equivalent number ofmock-transduced T cells, or no T cells. Mice from each group arerandomly bled for determination of peripheral blood CD19⁺ ALL blastcounts and then killed on days 35 and 49. The remaining animals areevaluated on days 57 and 70. Similar experiments can be done with CD123CARTS.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., at Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation isperformed in microtiter plates by mixing washed T cells with K562 cellsexpressing CD19 (K19) or CD32 and CD137 (KT32-BBL) for a finalT-cell:K562 ratio of 2:1. K562 cells are irradiated with gamma-radiationprior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonalantibodies are added to cultures with KT32-BBL cells to serve as apositive control for stimulating T-cell proliferation since thesesignals support long-term CD8⁺ T cell expansion ex vivo. T cells areenumerated in cultures using CountBright™ fluorescent beads (Invitrogen,Carlsbad, Calif.) and flow cytometry as described by the manufacturer.CAR⁺ T cells are identified by GFP expression using T cells that areengineered with eGFP-2A linked CAR-expressing lentiviral vectors. ForCAR+ T cells not expressing GFP, the CAR+ T cells are detected withbiotinylated recombinant CD123 protein and a secondary avidin-PEconjugate. CD4+ and CD8⁺ expression on T cells are also simultaneouslydetected with specific monoclonal antibodies (BD Biosciences). Cytokinemeasurements are performed on supernatants collected 24 hours followingre-stimulation using the human TH1/TH2 cytokine cytometric bead arraykit (BD Biosciences, San Diego, Calif.) according the manufacturer'sinstructions or using a Luminex 30-plex kit (Invitrogen). Fluorescenceis assessed using a BD Fortessa flow cytometer, and data is analyzedaccording to the manufacturer's instructions. Similar experiments can bedone with CD123 CARTS.

Cytotoxicity can be assessed by a standard 51Cr-release assay. See,e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly,target cells (K562 lines and primary pro-B-ALL cells) are loaded with51Cr (as NaCrO4, New England Nuclear, Boston, Mass.) at 37° C. for 2hours with frequent agitation, washed twice in complete RPMI and platedinto microtiter plates. Effector T cells are mixed with target cells inthe wells in complete RPMI at varying ratios of effector cell:targetcell (E:T). Additional wells containing media only (spontaneous release,SR) or a 1% solution of triton-X 100 detergent (total release, TR) arealso prepared. After 4 hours of incubation at 37° C., supernatant fromeach well is harvested. Released 51Cr is then measured using a gammaparticle counter (Packard Instrument Co., Waltham, Mass.). Eachcondition is performed in at least triplicate, and the percentage oflysis is calculated using the formula: % Lysis=(ER−SR)/(TR−SR), where ERrepresents the average 51Cr released for each experimental condition.

Imaging technologies can be used to evaluate specific trafficking andproliferation of CARs in tumor-bearing animal models. Such assays havebeen described, for example, in Barrett et al., Human Gene Therapy22:1575-1586 (2011). Briefly, NOD/SCID/γc^(−/−) (NSG) mice are injectedIV with Nalm-6 cells followed 7 days later with T cells 4 hour afterelectroporation with the CAR constructs. The T cells are stablytransfected with a lentiviral construct to express firefly luciferase,and mice are imaged for bioluminescence. Alternatively, therapeuticefficacy and specificity of a single injection of CAR⁺ T cells in Nalm-6xenograft model can be measured as the following: NSG mice are injectedwith Nalm-6 transduced to stably express firefly luciferase, followed bya single tail-vein injection of T cells electroporated with CD123 CAR 7days later. Animals are imaged at various time points post injection.For example, photon-density heat maps of firefly luciferasepositiveleukemia in representative mice at day 5 (2 days before treatment) andday 8 (24 hr post CAR⁺PBLs) can be generated.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate theCD123 CAR constructs of the invention.

Alternatively, or in combination to the methods disclosed herein,methods and compositions for one or more of: detection and/orquantification of CAR-expressing cells (e.g., in vitro or in vivo (e.g.,clinical monitoring)); immune cell expansion and/or activation; and/orCAR-specific selection, that involve the use of a CAR ligand, aredisclosed. In one exemplary embodiment, the CAR ligand is an antibodythat binds to the CAR molecule, e.g., binds to the extracellular antigenbinding domain of CAR (e.g., an antibody that binds to the antigenbinding domain, e.g., an anti-idiotypic antibody; or an antibody thatbinds to a constant region of the extracellular binding domain). Inother embodiments, the CAR ligand is a CAR antigen molecule (e.g., a CARantigen molecule as described herein).

In one aspect, a method for detecting and/or quantifying CAR-expressingcells is disclosed. For example, the CAR ligand can be used to detectand/or quantify CAR-expressing cells in vitro or in vivo (e.g., clinicalmonitoring of CAR-expressing cells in a patient, or dosing a patient).The method includes:

providing the CAR ligand (optionally, a labelled CAR ligand, e.g., a CARligand that includes a tag, a bead, a radioactive or fluorescent label);

acquiring the CAR-expressing cell (e.g., acquiring a sample containingCAR-expressing cells, such as a manufacturing sample or a clinicalsample);

contacting the CAR-expressing cell with the CAR ligand under conditionswhere binding occurs, thereby detecting the level (e.g., amount) of theCAR-expressing cells present. Binding of the CAR-expressing cell withthe CAR ligand can be detected using standard techniques such as FACS,ELISA and the like.

In another aspect, a method of expanding and/or activating cells (e.g.,immune effector cells) is disclosed. The method includes:

providing a CAR-expressing cell (e.g., a first CAR-expressing cell or atransiently expressing CAR cell);

contacting said CAR-expressing cell with a CAR ligand, e.g., a CARligand as described herein), under conditions where immune cellexpansion and/or proliferation occurs, thereby producing the activatedand/or expanded cell population.

In certain embodiments, the CAR ligand is present on (e.g., isimmobilized or attached to a substrate, e.g., a non-naturally occurringsubstrate). In some embodiments, the substrate is a non-cellularsubstrate. The non-cellular substrate can be a solid support chosenfrom, e.g., a plate (e.g., a microtiter plate), a membrane (e.g., anitrocellulose membrane), a matrix, a chip or a bead. In embodiments,the CAR ligand is present in the substrate (e.g., on the substratesurface). The CAR ligand can be immobilized, attached, or associatedcovalently or non-covalently (e.g., cross-linked) to the substrate. Inone embodiment, the CAR ligand is attached (e.g., covalently attached)to a bead. In the aforesaid embodiments, the immune cell population canbe expanded in vitro or ex vivo. The method can further includeculturing the population of immune cells in the presence of the ligandof the CAR molecule, e.g., using any of the methods described herein.

In other embodiments, the method of expanding and/or activating thecells further comprises addition of a second stimulatory molecule, e.g.,CD28. For example, the CAR ligand and the second stimulatory moleculecan be immobilized to a substrate, e.g., one or more beads, therebyproviding increased cell expansion and/or activation.

In yet another aspect, a method for selecting or enriching for a CARexpressing cell is provided. The method includes contacting the CARexpressing cell with a CAR ligand as described herein; and selecting thecell on the basis of binding of the CAR ligand.

In yet other embodiments, a method for depleting, reducing and/orkilling a CAR expressing cell is provided. The method includescontacting the CAR expressing cell with a CAR ligand as describedherein; and targeting the cell on the basis of binding of the CARligand, thereby reducing the number, and/or killing, the CAR-expressingcell. In one embodiment, the CAR ligand is coupled to a toxic agent(e.g., a toxin or a cell ablative drug). In another embodiment, theanti-idiotypic antibody can cause effector cell activity, e.g., ADCC orADC activities.

Exemplary anti-CAR antibodies that can be used in the methods disclosedherein are described, e.g., in WO 2014/190273 and by Jena et al.,“Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to DetectCD19-Specific T cells in Clinical Trials”, PLOS March 2013 8:3 e57838,the contents of which are incorporated by reference. In one embodiment,the anti-idiotypic antibody molecule recognizes an anti-CD19 antibodymolecule, e.g., an anti-CD19 scFv. For instance, the anti-idiotypicantibody molecule can compete for binding with the CD19-specific CAR mAbclone no. 136.20.1 described in Jena et al., PLOS March 2013 8:3 e57838;may have the same CDRs (e.g., one or more of, e.g., all of, VH CDR1, VHCDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3, using the Kabatdefinition, the Chothia definition, or a combination of the Kabat andChothia definitions) as the CD19-specific CAR mAb clone no. 136.20.1;may have one or more (e.g., 2) variable regions as the CD19-specific CARmAb clone no. 136.20.1, or may comprise the CD19-specific CAR mAb cloneno. 136.20.1. In some embodiments, the anti-idiotypic antibody was madeaccording to a method described in Jena et al. In another embodiment,the anti-idiotypic antibody molecule is an anti-idiotypic antibodymolecule described in WO 2014/190273. In some embodiments, theanti-idiotypic antibody molecule has the same CDRs (e.g., one or moreof, e.g., all of, VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VLCDR3) as an antibody molecule of WO 2014/190273 such as 136.20.1; mayhave one or more (e.g., 2) variable regions of an antibody molecule ofWO 2014/190273, or may comprise an antibody molecule of WO 2014/190273such as 136.20.1. In other embodiments, the anti-CAR antibody binds to aconstant region of the extracellular binding domain of the CAR molecule,e.g., as described in WO 2014/190273. In some embodiments, the anti-CARantibody binds to a constant region of the extracellular binding domainof the CAR molecule, e.g., a heavy chain constant region (e.g., aCH2-CH3 hinge region) or light chain constant region. For instance, insome embodiments the anti-CAR antibody competes for binding with the 2D3monoclonal antibody described in WO 2014/190273, has the same CDRs(e.g., one or more of, e.g., all of, VH CDR1, VH CDR2, CH CDR3, VL CDR1,VL CDR2, and VL CDR3) as 2D3, or has one or more (e.g., 2) variableregions of 2D3, or comprises 2D3 as described in WO 2014/190273.

In some aspects and embodiments, the compositions and methods herein areoptimized for a specific subset of T cells, e.g., as described in U.S.Ser. No. 62/031,699 filed Jul. 31, 2014, the contents of which areincorporated herein by reference in their entirety. In some embodiments,the optimized subsets of T cells display an enhanced persistencecompared to a control T cell, e.g., a T cell of a different type (e.g.,CD8⁺ or CD4⁺) expressing the same construct.

In some embodiments, a CD4⁺ T cell comprises a CAR described herein,which CAR comprises an intracellular signaling domain suitable for(e.g., optimized for, e.g., leading to enhanced persistence in) a CD4⁺ Tcell, e.g., an ICOS domain. In some embodiments, a CD8⁺ T cell comprisesa CAR described herein, which CAR comprises an intracellular signalingdomain suitable for (e.g., optimized for, e.g., leading to enhancedpersistence of) a CD8⁺ T cell, e.g., a 4-1BB domain, a CD28 domain, oranother costimulatory domain other than an ICOS domain. In someembodiments, the CAR described herein comprises an antigen bindingdomain described herein, e.g., a CAR comprising an antigen bindingdomain that specifically binds CD123, e.g., a CAR of Table 2 or Table 6.

In an aspect, described herein is a method of treating a subject, e.g.,a subject having cancer. The method includes administering to saidsubject, an effective amount of:

1) a CD4⁺ T cell comprising a CAR (the CAR^(CD4+))

comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein, e.g., an antigen binding domain that specifically binds CD123,e.g., an antigen-binding domain of Table 2, Table 6, or Table 9;

a transmembrane domain; and

an intracellular signaling domain, e.g., a first costimulatory domain,e.g., an ICOS domain; and

-   -   2) a CD8⁺ T cell comprising a CAR (the CAR^(CD8+)) comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein, e.g., an antigen binding domain that specifically binds CD123,e.g., an antigen-binding domain of Table 2, Table 6, or Table 9;

a transmembrane domain; and

an intracellular signaling domain, e.g., a second co stimulatory domain,e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domainother than an ICOS domain;

wherein the CAR^(CD4+) and the CAR^(CD8+) differ from one another.

Optionally, the method further includes administering:

3) a second CD8+ T cell comprising a CAR (the second CAR^(CD8+))comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein, e.g., an antigen binding domain that specifically binds CD123,e.g., an antigen-binding domain of Table 2, Table 6, or Table 9;

a transmembrane domain; and

an intracellular signaling domain, wherein the second CAR^(CD8+)comprises an intracellular signaling domain, e.g., a costimulatorysignaling domain, not present on the CAR^(CD8+), and, optionally, doesnot comprise an ICOS signaling domain.

Therapeutic Application

CD123 Associated Diseases and/or Disorders

The present invention provides, among other things, compositions andmethods for treating a disease associated with expression of CD123 orcondition associated with cells which express CD123 including, e.g., aproliferative disease such as a cancer or malignancy or a precancerouscondition such as a myelodysplasia, a myelodysplastic syndrome or apreleukemia; or a noncancer related indication associated with cellswhich express CD123. In one aspect, a cancer associated with expressionof CD123 is a hematological cancer. In one aspect, a hematologicalcancer includes but is not limited to AML, myelodysplastic syndrome,ALL, chronic myeloid leukemia, blastic plasmacytoid dendritic cellneoplasm, myeloproliferative neoplasms, Hodgkin lymphoma, and the like.Further disease associated with expression of CD123 expression include,but are not limited to, e.g., atypical and/or non-classical cancers,malignancies, precancerous conditions or proliferative diseasesassociated with expression of CD123. Non-cancer related indicationsassociated with expression of CD123 may also be included.

In one aspect, the invention provides methods for treating a diseaseassociated with CD123 expression. In one aspect, the invention providesmethods for treating a disease wherein part of the tumor is negative forCD123 and part of the tumor is positive for CD123. For example, the CARof the invention is useful for treating subjects that have undergonetreatment for a disease associated with elevated expression of CD123,wherein the subject that has undergone treatment for elevated levels ofCD123 exhibits a disease associated with elevated levels of CD123. Inembodiments, the CAR of the invention is useful for treating subjectsthat have undergone treatment for a disease associated with expressionof CD123, wherein the subject that has undergone treatment related toexpression of CD123 exhibits a disease associated with expression ofCD123.

In one aspect, the invention pertains to a vector comprising CD123 CARoperably linked to promoter for expression in mammalian immune effectorcells, e.g., T cells or NK cells. In one aspect, the invention providesa recombinant T cell expressing the CD123 CAR for use in treatingCD123-expressing tumors, wherein the recombinant immune effector cells,e.g., T cells or NK cells expressing the CD123 CAR is termed a CD123CAR-expressing cell (e.g., CD123 CART or CD123 CAR-expressing NK cell).In one aspect, the CD123 CART of the invention is capable of contactinga tumor cell with at least one CD123 CAR of the invention expressed onits surface such that the CD123 CAR-expressing cell (e.g., CD123 CART orCD123 CAR-expressing NK cell) targets the tumor cell and growth of thetumor is inhibited.

In one aspect, the invention pertains to a method of inhibiting growthof a CD123-expressing tumor cell, comprising contacting the tumor cellwith a CD123 CAR-expressing cell (e.g., CD123 CART or CD123CAR-expressing NK cell) of the present invention such that the CD123CAR-expressing cell (e.g., CD123 CART or CD123 CAR-expressing NK cell)is activated in response to the antigen and targets the cancer cell,wherein the growth of the tumor is inhibited.

In one aspect, the invention pertains to a method of treating cancer ina subject. The method comprises administering to the subject a CD123CAR-expressing cell (e.g., CD123 CART or CD123 CAR-expressing NK cell)of the present invention such that the cancer is treated in the subject.An example of a cancer that is treatable by the CD123 CAR-expressingcell (e.g., CD123 CART or CD123 CAR-expressing NK cell) of the inventionis a cancer associated with expression of CD123. An example of a cancerthat is treatable by the CD123 CAR-expressing cell (e.g., CD123 CART orCD123 CAR-expressing NK cell) of the invention includes but is notlimited to AML, Hodgkin lymphoma, myelodysplastic syndrome, chronicmyeloid leukemia and other myeloproliferative neoplasms, or Blasticplasmacytoid dendritic cell neoplasm, and the like.

The invention includes a type of cellular therapy where immune effectorcells, e.g., T cells or NK cells, are genetically modified to express achimeric antigen receptor (CAR) and the CD123 CAR-expressing cell (e.g.,CD123 CART or CD123 CAR-expressing NK cell) is infused to a recipient inneed thereof. The infused cell is able to kill tumor cells in therecipient. Unlike antibody therapies, CAR-modified immune effectorcells, e.g., the CAR-modified T cells or CAR-modified NK cells) are ableto replicate in vivo resulting in long-term persistence that can lead tosustained tumor control. In various aspects, the immune effector cells,e.g., T cells or NK cells, administered to the patient, or theirprogeny, persist in the patient for at least four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, twelve months, thirteen months, fourteen month, fifteen months,sixteen months, seventeen months, eighteen months, nineteen months,twenty months, twenty-one months, twenty-two months, twenty-threemonths, two years, three years, four years, or five years afteradministration of the immune effector cell, e.g., T cell or NK cell, tothe patient.

The invention also includes a type of cellular therapy where immuneeffector cells, e.g., T cells or NK cells, are modified, e.g., by invitro transcribed RNA, to transiently express a chimeric antigenreceptor (CAR) and the CAR-expressing cell, e.g., CAR T cell or CAR NKcell) is infused to a recipient in need thereof. The infused cell isable to kill tumor cells in the recipient. Thus, in various aspects, theimmune effector cells, e.g., T cells or NK cells, administered to thepatient, is present for less than one month, e.g., three weeks, twoweeks, one week, after administration of the immune effector cell, e.g.,T cell or NK cell, to the patient.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified immune effector cells,e.g., T cells or NK cells, may be an active or a passive immuneresponse, or alternatively may be due to a direct vs indirect immuneresponse. In one aspect, the CAR transduced immune effector cells, e.g.,T cells or NK cells, exhibit specific proinflammatory cytokine secretionand potent cytolytic activity in response to human cancer cellsexpressing CD123, resist soluble CD123 inhibition, mediate bystanderkilling and mediate regression of an established human tumor. Forexample, antigen-less tumor cells within a heterogeneous field ofCD123-expressing tumor may be susceptible to indirect destruction byCD123-redirected immune effector cell, e.g., T cells or NK cells, thathas previously reacted against adjacent antigen-positive cancer cells.

In one aspect, the fully-human CAR-modified immune effector cells (e.g.,T cells or NK cells) of the invention may be a type of vaccine for exvivo immunization and/or in vivo therapy in a mammal. In one aspect, themammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell, e.g., T cell or NKcell, into a mammal: i) expansion of the cells, ii) introducing anucleic acid encoding a CAR to the cells or iii) cryopreservation of thecells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modifiedimmune effector cells (e.g., T cells or NK cells) of the invention areused in the treatment of diseases, disorders and conditions associatedwith expression of CD123. In certain aspects, the cells of the inventionare used in the treatment of patients at risk for developing diseases,disorders and conditions associated with expression of CD123. Thus, thepresent invention provides methods for the treatment or prevention ofdiseases, disorders and conditions associated with expression of CD123comprising administering to a subject in need thereof, a therapeuticallyeffective amount of the CAR-modified immune effector cells (e.g., Tcells or NK cells) of the invention.

In one aspect, the CAR-expressing cells (CART cells or CAR-expressing NKcells) of the inventions may be used to treat a proliferative diseasesuch as a cancer or malignancy or is a precancerous condition such as amyelodysplasia, a myelodysplastic syndrome or a preleukemia. In oneaspect, a cancer associated with expression of CD123 is a hematologicalcancer preleukemia, a hyperproliferative disorder, a hyperplasia or adysplasia, which is characterized by abnormal growth of cells.

In one aspect, the CAR-expressing cells (CART cells or CAR-expressing NKcells) of the invention are used to treat a cancer, wherein the canceris a hematological cancer. Hematological cancer conditions are the typesof cancer such as leukemia and malignant lymphoproliferative conditionsthat affect blood, bone marrow and the lymphatic system.

In one aspect, the compositions and CAR-expressing cells (CART cells orCAR-expressing NK cells) of the present invention are particularlyuseful for treating myeloid leukemias, AML and its subtypes, chronicmyeloid leukemia (CML), myelodysplastic syndrome (MDS),myeloproliferative neoplasms (MPN), histiocytic disorders, and mast celldisorders.

Leukemia can be classified as acute leukemia and chronic leukemia. Acuteleukemia can be further classified as acute myelogenous leukemia (AML)and acute lymphoid leukemia (ALL). Chronic leukemia includes chronicmyelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Otherrelated conditions include myelodysplastic syndromes (MDS, formerlyknown as “preleukemia”) which are a diverse collection of hematologicalconditions united by ineffective production (or dysplasia) of myeloidblood cells and risk of transformation to AML.

Lymphoma is a group of blood cell tumors that develop from lymphocytes.Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

In AML, malignant transformation and uncontrolled proliferation of anabnormally differentiated, long-lived myeloid progenitor cell results inhigh circulating numbers of immature blood forms and replacement ofnormal marrow by malignant cells. Symptoms include fatigue, pallor, easybruising and bleeding, fever, and infection; symptoms of leukemicinfiltration are present in only about 5% of patients (often as skinmanifestations). Examination of peripheral blood smear and bone marrowis diagnostic. Existing treatment includes induction chemotherapy toachieve remission and post-remission chemotherapy (with or without stemcell transplantation) to avoid relapse.

AML has a number of subtypes that are distinguished from each other bymorphology, immunophenotype, and cytochemistry. Five classes aredescribed, based on predominant cell type, including myeloid,myeloid-monocytic, monocytic, erythroid, and megakaryocytic.

Remission induction rates range from 50 to 85%. Long-term disease-freesurvival reportedly occurs in 20 to 40% of patients and increases to 40to 50% in younger patients treated with stem cell transplantation.

Prognostic factors help determine treatment protocol and intensity;patients with strongly negative prognostic features are usually givenmore intense forms of therapy, because the potential benefits arethought to justify the increased treatment toxicity. The most importantprognostic factor is the leukemia cell karyotype; favorable karyotypesinclude t(15;17), t(8;21), and inv16 (p13;q22). Negative factors includeincreasing age, a preceding myelodysplastic phase, secondary leukemia,high WBC count, and absence of Auer rods.

Initial therapy attempts to induce remission and differs most from ALLin that AML responds to fewer drugs. The basic induction regimenincludes cytarabine by continuous IV infusion or high doses for 5 to 7days; daunorubicin or idarubicin is given IV for 3 days during thistime. Some regimens include 6-thioguanine, etoposide, vincristine, andprednisone, but their contribution is unclear. Treatment usually resultsin significant myelosuppression, with infection or bleeding; there issignificant latency before marrow recovery. During this time, meticulouspreventive and supportive care is vital.

Chronic myelogenous (or myeloid) leukemia (CML) is also known as chronicgranulocytic leukemia, and is characterized as a cancer of the whiteblood cells. Common treatment regimens for CML include Bcr-Abl tyrosinekinase inhibitors, imatinib (Gleevec®), dasatinib and nilotinib. Bcr-Abltyrosine kinase inhibitors are specifically useful for CML patients withthe Philadelphia chromosome translocation.

Myelodysplastic syndromes (MDS) are hematological medical conditionscharacterized by disorderly and ineffective hematopoiesis, or bloodproduction. Thus, the number and quality of blood-forming cells declineirreversibly. Some patients with MDS can develop severe anemia, whileothers are asymptomatic. The classification scheme for MDS is known inthe art, with criteria designating the ratio or frequency of particularblood cell types, e.g., myeloblasts, monocytes, and red cell precursors.MDS includes refractory anemia, refractory anemia with ringsideroblasts, refractory anemia with excess blasts, refractory anemiawith excess blasts in transformation, chronic myelomonocytic leukemia(CML).

Treatments for MDS vary with the severity of the symptoms. Aggressiveforms of treatment for patients experiencing severe symptoms includebone marrow transplants and supportive care with blood product support(e.g., blood transfusions) and hematopoietic growth factors (e.g.,erythropoietin). Other agents are frequently used to treat MDS:5-azacytidine, decitabine, and lenalidomide. In some cases, ironchelators deferoxamine (Desferal®) and deferasirox (Exjade®) may also beadministered.

In another embodiment, the CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) of the present invention are used to treatcancers or leukemias with leukemia stem cells. For example, the leukemiastem cells are CD34⁺/CD38⁻ leukemia cells.

The present invention provides, among other things, compositions andmethods for treating cancer. In one aspect, the cancer is a hematologiccancer including but is not limited to leukemia (such as acutemyelogenous leukemia, chronic myelogenous leukemia, acute lymphoidleukemia, chronic lymphoid leukemia and myelodysplastic syndrome) andmalignant lymphoproliferative conditions, including lymphoma (such asmultiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt'slymphoma, and small cell- and large cell-follicular lymphoma).

In one aspect, the CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) of the invention may be used to treat othercancers and malignancies such as, but not limited to, e.g., acuteleukemias including but not limited to, e.g., B-cell acute lymphoidleukemia (“B-ALL”), T-cell acute lymphoid leukemia (“T-ALL”), acutelymphoid leukemia (ALL); one or more chronic leukemias including but notlimited to, e.g., chronic myelogenous leukemia (CML), chroniclymphocytic leukemia (CLL); additional hematologic cancers orhematologic conditions including, but not limited to, e.g., B cellprolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm,Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma,Hairy cell leukemia, small cell- or a large cell-follicular lymphoma,malignant lymphoproliferative conditions, MALT lymphoma, mantle celllymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia, and “preleukemia” which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells, and the like The CAR-modifiedimmune effector cells, e.g., T cells or NK cells, of the presentinvention may be administered either alone, or as a pharmaceuticalcomposition in combination with diluents and/or with other componentssuch as IL-2 or other cytokines or cell populations.

The present invention also provides methods for inhibiting theproliferation or reducing a CD123-expressing cell population, themethods comprising contacting a population of cells comprising aCD123-expressing cell with a CAR-expressing cell (e.g., CD123 CART cellor CD123 CAR-expressing NK cell) of the invention that binds to theCD123-expressing cell. In a specific aspect, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing CD123, the methods comprisingcontacting the CD123-expressing cancer cell population with aCAR-expressing cell (e.g., CD123 CART cell or CD123 CAR-expressing NKcell) of the invention that binds to the CD123-expressing cell. In oneaspect, the present invention provides methods for inhibiting theproliferation or reducing the population of cancer cells expressingCD123, the methods comprising contacting the CD123-expressing cancercell population with a CAR-expressing cell (e.g., CD123 CART cell orCD123 CAR-expressing NK cell) of the invention that binds to theCD123-expressing cell. In certain aspects, the CAR-expressing cell(e.g., CD123 CART cell or CD123 CAR-expressing NK cell) of the inventionreduces the quantity, number, amount or percentage of cells and/orcancer cells by at least 25%, at least 30%, at least 40%, at least 50%,at least 65%, at least 75%, at least 85%, at least 95%, or at least 99%in a subject with or animal model for myeloid leukemia or another cancerassociated with CD123-expressing cells relative to a negative control.In one aspect, the subject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disease associated with CD123-expressing cells (e.g.,a hematologic cancer or atypical cancer expressing CD123), the methodscomprising administering to a subject in need a CAR-expressing cell(e.g., CD123 CART cell or CD123 CAR-expressing NK cell) of the inventionthat binds to the CD123-expressing cell. In one aspect, the subject is ahuman. Non-limiting examples of disorders associated withCD123-expressing cells include autoimmune disorders (such as lupus),inflammatory disorders (such as allergies and asthma) and cancers (suchas hematological cancers or atypical cancers expressing CD123).

The present invention also provides methods for preventing, treatingand/or managing a disease associated with CD123-expressing cells, themethods comprising administering to a subject in need a CAR-expressingcell (e.g., CD123 CART cell or CD123 CAR-expressing NK cell) of theinvention that binds to the CD123-expressing cell. In one aspect, thesubject is a human.

The present invention provides methods for preventing relapse of cancerassociated with CD123-expressing cells, the methods comprisingadministering to a subject in need thereof a CAR-expressing cell (e.g.,CD123 CART cell or CD123 CAR-expressing NK cell) of the invention thatbinds to the CD123-expressing cell. In one aspect, the methods compriseadministering to the subject in need thereof an effective amount of aCAR-expressing cell (e.g., CD123 CART cell or CD123 CAR-expressing NKcell) described herein that binds to the CD123-expressing cell incombination with an effective amount of another therapy.

Bone Marrow Ablation

In one aspect, the present invention provides compositions and methodsfor bone marrow ablation. For example, in one aspect, the inventionprovides compositions and methods for eradication of at least a portionof existing bone marrow in a subject. It is described herein that, incertain instances, the CART123 cells comprising a CD123 CAR of thepresent invention eradicates CD123 positive bone marrow myeloidprogenitor cells.

In one aspect, the invention provides a method of bone marrow ablationcomprising administering a CAR-expressing cell (e.g., CD123 CART cell orCD123 CAR-expressing NK cell) of the invention to a subject in need ofbone marrow ablation. For example, the present method may be used toeradicate some or all of the existing bone marrow of a subject having adisease or disorder in which bone marrow transplantation or bone marrowreconditioning is a beneficial treatment strategy. In one aspect, thebone marrow ablation method of the invention, comprising theadministration of a CAR-expressing cell (e.g., CD123 CART cell or CD123CAR-expressing NK cell) described elsewhere herein, is performed in asubject prior to bone marrow transplantation. Thus, in one aspect, themethod of the invention provides a cellular conditioning regimen priorto bone marrow or stem cell transplantation. In one aspect, bone marrowtransplantation comprises transplantation of a stem cell. The bonemarrow transplantation may comprise transplantation of autologous orallogeneic cells.

The present invention provides a method of treating a disease ordisorder comprising administering a CAR-expressing cell (e.g., CD123CART cell or CD123 CAR-expressing NK cell) of the invention to eradicateat least a portion of existing bone marrow. The method may be used as atleast a portion of a treatment regimen for treating any disease ordisorder where bone marrow transplantation is beneficial. That is, thepresent method may be used in any subject in need of a bone marrowtransplant. In one aspect, bone marrow ablation comprisingadministration of a CAR-expressing cell (e.g., CD123 CART cell or CD123CAR-expressing NK cell) is useful in the treatment of AML. In certainaspects, bone marrow ablation by way of the present method is useful intreating a hematological cancer, a solid tumor, a hematologic disease, ametabolic disorder, HIV, HTLV, a lysosomal storage disorder, and animmunodeficiency.

Compositions and methods disclosed herein may be used to eradicate atleast a portion of existing bone marrow to treat hematological cancersincluding, but not limited to, leukemia, lymphoma, myeloma, ALL, AML,CLL, CML, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and multiplemyeloma.

Compositions and methods disclosed herein may be used to treathematologic diseases including, but not limited to myelodysplasia,anemia, paroxysmal nocturnal hemoglobinuria, aplastic anemia, acquiredpure red cell anemia, Diamon-Blackfan anemia, Fanconi anemia, cytopenia,amegakaryotic thrombocytopenia, myeloproliferative disorders,polycythemia vera, essential thrombocytosis, myelofibrosis,hemoglobinopathies, sickle cell disease, 0 thalassemia major, amongothers.

Compositions and methods disclosed herein may be used to treat lysosomalstorage disorders including, but not limited to lipidoses,sphigolipodeses, leukodystrophies, mucopolysaccharidoses,glycoproteinoses, infantile neuronal ceroid lipofuscinosis,Jansky-Bielschowsky disease, Niemann-Pick disease, Gaucher disease,adrenoleukodystrophy, metachromatic leukodystrophy, Krabbe disease,Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, huntersyndrome, Sanfilippo syndrome, Morquio syndrome, Maroteaux-Lamysyndrome, Sly syndrome, mucolipidosis, fucolipidosis,aspartylglucosaminuria, alpha-mannosidoses, and Wolman disease.

Compositions and methods disclosed herein may be used to treatimmunodeficiencies including, but not limited to, T-cell deficiencies,combined T-cell and B-cell deficiencies, phagocyte disorders, immunedysregulation diseases, innate immune deficiencies, ataxiatelangiectasia, DiGeorge syndrome, severe combined immunodeficiency(SCID), Wiskott-Aldrich syndrome, Kostmann syndrome, Shwachman-Diamondsyndrome, Griscelli syndrome, and NF-Kappa-B Essential Modulator (NEMO)deficiency.

In one aspect, the present invention provides a method of treatingcancer comprising bone marrow conditioning, where at least a portion ofbone marrow of the subject is eradicated by the CAR-expressing cell(e.g., CD123 CART cell or CD123 CAR-expressing NK cell) of theinvention. For example, in certain instances, the bone marrow of thesubject comprises a malignant precursor cell that can be targeted andeliminated by the activity of the CAR-expressing cell (e.g., CD123 CARTcell or CD123 CAR-expressing NK cell). In one aspect, a bone marrowconditioning therapy comprises administering a bone marrow or stem celltransplant to the subject following the eradication of native bonemarrow. In one aspect, the bone marrow reconditioning therapy iscombined with one or more other anti-cancer therapies, including, butnot limited to anti-tumor CAR therapies, chemotherapy, radiation, andthe like.

In one aspect, eradication of the administered CAR-expressing cell(e.g., CD123 CART cell or CD123 CAR-expressing NK cell) may be requiredprior to infusion of bone marrow or stem cell transplant. Eradication ofthe CAR-expressing cell (e.g., CD123 CART cell or CD123 CAR-expressingNK cell) may be accomplished using any suitable strategy or treatment,including, but not limited to, use of a suicide gene, limited CARpersistence using RNA encoded CARs, or anti-T cell modalities includingantibodies or chemotherapy.

Combination Therapies

A CAR-expressing cell described herein may be used in combination withother known agents and therapies. Administered “in combination”, as usedherein, means that two (or more) different treatments are delivered tothe subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

A CAR-expressing cell described herein and the at least one additionaltherapeutic agent can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the CAR-expressing cell described herein can be administered first, andthe additional agent can be administered second, or the order ofadministration can be reversed.

The CAR therapy and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The CAR therapy canbe administered before the other treatment, concurrently with thetreatment, post-treatment, or during remission of the disorder.

When administered in combination, the CAR therapy and the additionalagent (e.g., second or third agent), or all, can be administered in anamount or dose that is higher, lower or the same than the amount ordosage of each agent used individually, e.g., as a monotherapy. Incertain embodiments, the administered amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all, islower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%)than the amount or dosage of each agent used individually, e.g., as amonotherapy. In other embodiments, the amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all,that results in a desired effect (e.g., treatment of cancer) is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower)than the amount or dosage of each agent used individually, e.g., as amonotherapy, required to achieve the same therapeutic effect.

In further aspects, a CAR-expressing cell described herein may be usedin a treatment regimen in combination with surgery, cytokines,radiation, or chemotherapy such as cytoxan, fludarabine, histonedeacetylase inhibitors, demethylating agents, or peptide vaccine, suchas that described in Izumoto et al. 2008 J Neurosurg 108:963-971.

In certain instances, compounds of the present invention are combinedwith other therapeutic agents, such as other anti-cancer agents,anti-allergic agents, anti-nausea agents (or anti-emetics), painrelievers, cytoprotective agents, and combinations thereof.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include an anthracycline (e.g., doxorubicin (e.g., liposomaldoxorubicin)). a vinca alkaloid (e.g., vinblastine, vincristine,vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide,decarbazine, melphalan, ifosfamide, temozolomide), an immune cellantibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab,tositumomab, brentuximab), an antimetabolite (including, e.g., folicacid antagonists, pyrimidine analogs, purine analogs and adenosinedeaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFRglucocorticoid induced TNFR related protein (GITR) agonist, a proteasomeinhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), animmunomodulator such as thalidomide or a thalidomide derivative (e.g.,lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Anti-cancer agents of particular interest for combinations with thecompounds of the present invention include: anthracyclines; alkylatingagents; antimetabolites; drugs that inhibit either the calcium dependentphosphatase calcineurin or the p70S6 kinase FK506) or inhibit the p70S6kinase; mTOR inhibitors; immunomodulators; anthracyclines; vincaalkaloids; proteosome inhibitors; GITR agonists; protein tyrosinephosphatase inhibitors; a CDK4 kinase inhibitor; a BTK inhibitor; a MKNkinase inhibitor; a DGK kinase inhibitor; or an oncolytic virus.

Exemplary antimetabolites include, without limitation, pyrimidineanalogs, purine analogs and adenosine deaminase inhibitors):methotrexate (Rheumatrex®, Trexall®), 5-fluorouracil (Adrucil®, Efudex®,Fluoroplex®), floxuridine (FUDF®), cytarabine (Cytosar-U®, TarabinePFS), 6-mercaptopurine (Puri-Nethol®)), 6-thioguanine (ThioguanineTabloid®), fludarabine phosphate (Fludara®), pentostatin (Nipent®),pemetrexed (Alimta®), raltitrexed (Tomudex®), cladribine (Leustatin®),clofarabine (Clofarex®, Clolar®), azacitidine (Vidaza®), decitabine andgemcitabine (Gemzar®). Preferred antimetabolites include, cytarabine,clofarabine and fludarabine.

Exemplary alkylating agents include, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®,Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracilnitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®,Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®,Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide(Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman(Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®),triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa(Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®),lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine(DTIC-Dome®). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® andTemodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard,Alkeran®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan(Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (alsoknown as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® andPlatinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® andNeosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine(HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine(Matulane®); Mechlorethamine (also known as nitrogen mustard, mustineand mechloroethamine hydrochloride, Mustargen®); Streptozocin(Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA,Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®,Revimmune®); and Bendamustine HCl (Treanda®).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with fludarabine, cyclophosphamide, and/orrituximab. In embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with fludarabine,cyclophosphamide, and rituximab (FCR). In embodiments, the subject hasCLL. For example, the subject has a deletion in the short arm ofchromosome 17 (del(17p), e.g., in a leukemic cell). In other examples,the subject does not have a del(17p). In embodiments, the subjectcomprises a leukemic cell comprising a mutation in the immunoglobulinheavy-chain variable-region (IgV_(H)) gene. In other embodiments, thesubject does not comprise a leukemic cell comprising a mutation in theimmunoglobulin heavy-chain variable-region (IgV_(H)) gene. Inembodiments, the fludarabine is administered at a dosage of about 10-50mg/m² (e.g., about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or45-50 mg/m²), e.g., intravenously. In embodiments, the cyclophosphamideis administered at a dosage of about 200-300 mg/m² (e.g., about 200-225,225-250, 250-275, or 275-300 mg/m²), e.g., intravenously. Inembodiments, the rituximab is administered at a dosage of about 400-600mg/m2 (e.g., 400-450, 450-500, 500-550, or 550-600 mg/m²), e.g.,intravenously.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with bendamustine and rituximab. Inembodiments, the subject has CLL. For example, the subject has adeletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject comprises a leukemic cell comprising amutation in the immunoglobulin heavy-chain variable-region (IgV_(H))gene. In other embodiments, the subject does not comprise a leukemiccell comprising a mutation in the immunoglobulin heavy-chainvariable-region (IgV_(H)) gene. In embodiments, the bendamustine isadministered at a dosage of about 70-110 mg/m2 (e.g., 70-80, 80-90,90-100, or 100-110 mg/m2), e.g., intravenously. In embodiments, therituximab is administered at a dosage of about 400-600 mg/m2 (e.g.,400-450, 450-500, 500-550, or 550-600 mg/m²), e.g., intravenously.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab, cyclophosphamide,doxorubicine, vincristine, and/or a corticosteroid (e.g., prednisone).In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab, cyclophosphamide,doxorubicine, vincristine, and prednisone (R-CHOP). In embodiments, thesubject has diffuse large B-cell lymphoma (DLBCL). In embodiments, thesubject has nonbulky limited-stage DLBCL (e.g., comprises a tumor havinga size/diameter of less than 7 cm). In embodiments, the subject istreated with radiation in combination with the R-CHOP. For example, thesubject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5,or 6 cycles of R-CHOP), followed by radiation. In some cases, thesubject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5,or 6 cycles of R-CHOP) following radiation.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with etoposide, prednisone, vincristine,cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, aCAR-expressing cell described herein is administered to a subject incombination with etoposide, prednisone, vincristine, cyclophosphamide,doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressingcell described herein is administered to a subject in combination withdose-adjusted EPOCH-R (DA-EPOCH-R). In embodiments, the subject has a Bcell lymphoma, e.g., a Myc-rearranged aggressive B cell lymphoma.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab and/or lenalidomide.Lenalidomide ((RS)-3-(4-Amino-1-oxo1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione) is animmunomodulator. In embodiments, a CAR-expressing cell described hereinis administered to a subject in combination with rituximab andlenalidomide. In embodiments, the subject has follicular lymphoma (FL)or mantle cell lymphoma (MCL). In embodiments, the subject has FL andhas not previously been treated with a cancer therapy. In embodiments,lenalidomide is administered at a dosage of about 10-20 mg (e.g., 10-15or 15-20 mg), e.g., daily. In embodiments, rituximab is administered ata dosage of about 350-550 mg/m² (e.g., 350-375, 375-400, 400-425,425-450, 450-475, or 475-500 mg/m²), e.g., intravenously.

Exemplary mTOR inhibitors include, e.g., temsirolimus; ridaforolimus(formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus,(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 706), inner salt (SF1126, CAS 936487-67-1), and XL765.

Exemplary immunomodulators include, e.g., afutuzumab (available fromRoche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics).

Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® andRubex®); bleomycin (Lenoxane®); daunorubicin (dauorubicin hydrochloride,daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicinliposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone(DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®,Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin;ravidomycin; and desacetylravidomycin.

Exemplary vinca alkaloids include, e.g., vinorelbine tartrate(Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®));vinblastine (also known as vinblastine sulfate, vincaleukoblastine andVLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteosome inhibitors include bortezomib (Velcade®);carfilzomib (PX-171-007,(S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide);marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib(CEP-18770); andO-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide(ONX-0912).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with brentuximab. Brentuximab is anantibody-drug conjugate of anti-CD30 antibody and monomethyl auristatinE. In embodiments, the subject has Hodgkin's lymphoma (HL), e.g.,relapsed or refractory HL. In embodiments, the subject comprises CD30+HL. In embodiments, the subject has undergone an autologous stem celltransplant (ASCT). In embodiments, the subject has not undergone anASCT. In embodiments, brentuximab is administered at a dosage of about1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g.,intravenously, e.g., every 3 weeks.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with brentuximab and dacarbazine or incombination with brentuximab and bendamustine. Dacarbazine is analkylating agent with a chemical name of5-(3,3-Dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is analkylating agent with a chemical name of4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid.In embodiments, the subject has Hodgkin's lymphoma (HL). In embodiments,the subject has not previously been treated with a cancer therapy. Inembodiments, the subject is at least 60 years of age, e.g., 60, 65, 70,75, 80, 85, or older. In embodiments, dacarbazine is administered at adosage of about 300-450 mg/m² (e.g., about 300-325, 325-350, 350-375,375-400, 400-425, or 425-450 mg/m²), e.g., intravenously. Inembodiments, bendamustine is administered at a dosage of about 75-125mg/m2 (e.g., 75-100 or 100-125 mg/m², e.g., about 90 mg/m²), e.g.,intravenously. In embodiments, brentuximab is administered at a dosageof about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg),e.g., intravenously, e.g., every 3 weeks.

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a CD20 inhibitor, e.g., ananti-CD20 antibody (e.g., an anti-CD20 mono- or bispecific antibody) ora fragment thereof. Exemplary anti-CD20 antibodies include but are notlimited to rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab,TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921(Genentech). See, e.g., Lim et al. Haematologica. 95.1(2010):135-43.

In some embodiments, the anti-CD20 antibody comprises rituximab.Rituximab is a chimeric mouse/human monoclonal antibody IgG1 kappa thatbinds to CD20 and causes cytolysis of a CD20 expressing cell, e.g., asdescribed inwww.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf. Inembodiments, a CAR-expressing cell described herein is administered to asubject in combination with rituximab. In embodiments, the subject hasCLL or SLL.

In some embodiments, rituximab is administered intravenously, e.g., asan intravenous infusion. For example, each infusion provides about500-2000 mg (e.g., about 500-550, 550-600, 600-650, 650-700, 700-750,750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200,1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,1800-1900, or 1900-2000 mg) of rituximab. In some embodiments, rituximabis administered at a dose of 150 mg/m² to 750 mg/m², e.g., about 150-175mg/m², 175-200 mg/m², 200-225 mg/m², 225-250 mg/m², 250-300 mg/m²,300-325 mg/m², 325-350 mg/m², 350-375 mg/m², 375-400 mg/m², 400-425mg/m², 425-450 mg/m², 450-475 mg/m², 475-500 mg/m², 500-525 mg/m²,525-550 mg/m², 550-575 mg/m², 575-600 mg/m², 600-625 mg/m², 625-650mg/m², 650-675 mg/m², or 675-700 mg/m², where m² indicates the bodysurface area of the subject. In some embodiments, rituximab isadministered at a dosing interval of at least 4 days, e.g., 4, 7, 14,21, 28, 35 days, or more. For example, rituximab is administered at adosing interval of at least 0.5 weeks, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8weeks, or more. In some embodiments, rituximab is administered at a doseand dosing interval described herein for a period of time, e.g., atleast 2 weeks, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 weeks, or greater. For example, rituximab isadministered at a dose and dosing interval described herein for a totalof at least 4 doses per treatment cycle (e.g., at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, or more doses per treatment cycle).

In some embodiments, the anti-CD20 antibody comprises ofatumumab.Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody with amolecular weight of approximately 149 kDa. For example, ofatumumab isgenerated using transgenic mouse and hybridoma technology and isexpressed and purified from a recombinant murine cell line (NSO). See,e.g., www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf;and Clinical Trial Identifier number NCT01363128, NCT01515176,NCT01626352, and NCT01397591. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withofatumumab. In embodiments, the subject has CLL or SLL.

In some embodiments, ofatumumab is administered as an intravenousinfusion. For example, each infusion provides about 150-3000 mg (e.g.,about 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900,900-950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800,1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, or 2800-3000 mg)of ofatumumab. In embodiments, ofatumumab is administered at a startingdosage of about 300 mg, followed by 2000 mg, e.g., for about 11 doses,e.g., for 24 weeks. In some embodiments, ofatumumab is administered at adosing interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, ormore. For example, ofatumumab is administered at a dosing interval of atleast 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28,20, 22, 24, 26, 28, 30 weeks, or more. In some embodiments, ofatumumabis administered at a dose and dosing interval described herein for aperiod of time, e.g., at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50,60 weeks or greater, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months orgreater, or 1, 2, 3, 4, 5 years or greater. For example, ofatumumab isadministered at a dose and dosing interval described herein for a totalof at least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, or more doses per treatmentcycle).

In some cases, the anti-CD20 antibody comprises ocrelizumab. Ocrelizumabis a humanized anti-CD20 monoclonal antibody, e.g., as described inClinical Trials Identifier Nos. NCT00077870, NCT01412333, NCT00779220,NCT00673920, NCT01194570, and Kappos et al. Lancet.19.378(2011):1779-87.

In some cases, the anti-CD20 antibody comprises veltuzumab. Veltuzumabis a humanized monoclonal antibody against CD20. See, e.g., ClinicalTrial Identifier No. NCT00547066, NCT00546793, NCT01101581, andGoldenberg et al. Leuk Lymphoma. 51(5)(2010):747-55.

In some cases, the anti-CD20 antibody comprises GA101. GA101 (alsocalled obinutuzumab or RO5072759) is a humanized and glyco-engineeredanti-CD20 monoclonal antibody. See, e.g., Robak. Curr. Opin. Investig.Drugs. 10.6(2009):588-96; Clinical Trial Identifier Numbers:NCT01995669, NCT01889797, NCT02229422, and NCT01414205; andwww.accessdatafda.gov/drugsatfda_docs/label/2013/125486s000lbl.pdf.

In some cases, the anti-CD20 antibody comprises AME-133v. AME-133v (alsocalled LY2469298 or ocaratuzumab) is a humanized IgG1 monoclonalantibody against CD20 with increased affinity for the FcγRIIIa receptorand an enhanced antibody dependent cellular cytotoxicity (ADCC) activitycompared with rituximab. See, e.g., Robak et al. BioDrugs25.1(2011):13-25; and Forero-Torres et al. Clin Cancer Res.18.5(2012):1395-403.

In some cases, the anti-CD20 antibody comprises PRO131921. PRO131921 isa humanized anti-CD20 monoclonal antibody engineered to have betterbinding to FcγRIIIa and enhanced ADCC compared with rituximab. See,e.g., Robak et al. BioDrugs 25.1(2011):13-25; and Casulo et al. ClinImmunol. 154.1(2014):37-46; and Clinical Trial Identifier No.NCT00452127.

In some cases, the anti-CD20 antibody comprises TRU-015. TRU-015 is ananti-CD20 fusion protein derived from domains of an antibody againstCD20. TRU-015 is smaller than monoclonal antibodies, but retainsFc-mediated effector functions. See, e.g., Robak et al. BioDrugs25.1(2011):13-25. TRU-015 contains an anti-CD20 single-chain variablefragment (scFv) linked to human IgG1 hinge, CH2, and CH3 domains butlacks CH1 and CL domains.

In some embodiments, an anti-CD20 antibody described herein isconjugated or otherwise bound to a therapeutic agent, e.g., achemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylaseinhibitor, demethylating agent, peptide vaccine, anti-tumor antibiotic,tyrosine kinase inhibitor, alkylating agent, anti-microtubule oranti-mitotic agent), anti-allergic agent, anti-nausea agent (oranti-emetic), pain reliever, or cytoprotective agent described herein.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a B-cell lymphoma 2 (BCL-2) inhibitor(e.g., venetoclax, also called ABT-199 or GDC-0199) and/or rituximab. Inembodiments, a CAR-expressing cell described herein is administered to asubject in combination with venetoclax and rituximab. Venetoclax is asmall molecule that inhibits the anti-apoptotic protein, BCL-2. Thestructure of venetoclax(4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide)is shown below.

In embodiments, the subject has CLL. In embodiments, the subject hasrelapsed CLL, e.g., the subject has previously been administered acancer therapy. In embodiments, venetoclax is administered at a dosageof about 15-600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300,300-400, 400-500, or 500-600 mg), e.g., daily. In embodiments, rituximabis administered at a dosage of about 350-550 mg/m2 (e.g., 350-375,375-400, 400-425, 425-450, 450-475, or 475-500 mg/m2), e.g.,intravenously, e.g., monthly.

In some embodiments, a CAR-expressing cell described herein isadministered in combination with an oncolytic virus. In embodiments,oncolytic viruses are capable of selectively replicating in andtriggering the death of or slowing the growth of a cancer cell. In somecases, oncolytic viruses have no effect or a minimal effect onnon-cancer cells. An oncolytic virus includes but is not limited to anoncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolyticretrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolyticSinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g.,oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolyticmeasles virus, or oncolytic vesicular stomatitis virus (VSV)).

In some embodiments, the oncolytic virus is a virus, e.g., recombinantoncolytic virus, described in US2010/0178684 A1, which is incorporatedherein by reference in its entirety. In some embodiments, a recombinantoncolytic virus comprises a nucleic acid sequence (e.g., heterologousnucleic acid sequence) encoding an inhibitor of an immune orinflammatory response, e.g., as described in US2010/0178684 A1,incorporated herein by reference in its entirety. In embodiments, therecombinant oncolytic virus, e.g., oncolytic NDV, comprises apro-apoptotic protein (e.g., apoptin), a cytokine (e.g., GM-CSF,interferon-gamma, interleukin-2 (IL-2), tumor necrosis factor-alpha), animmunoglobulin (e.g., an antibody against ED-B firbonectin), tumorassociated antigen, a bispecific adapter protein (e.g., bispecificantibody or antibody fragment directed against NDV HN protein and a Tcell co-stimulatory receptor, such as CD3 or CD28; or fusion proteinbetween human IL-2 and single chain antibody directed against NDV HNprotein). See, e.g., Zamarin et al. Future Microbiol. 7.3(2012):347-67,incorporated herein by reference in its entirety. In some embodiments,the oncolytic virus is a chimeric oncolytic NDV described in U.S. Pat.No. 8,591,881 B2, US 2012/0122185 A1, or US 2014/0271677 A1, each ofwhich is incorporated herein by reference in their entireties.

In some embodiments, the oncolytic virus comprises a conditionallyreplicative adenovirus (CRAd), which is designed to replicateexclusively in cancer cells. See, e.g., Alemany et al. NatureBiotechnol. 18(2000):723-27. In some embodiments, an oncolyticadenovirus comprises one described in Table 1 on page 725 of Alemany etal., incorporated herein by reference in its entirety.

Exemplary oncolytic viruses include but are not limited to thefollowing:

Group B Oncolytic Adenovirus (ColoAd1) (PsiOxus Therapeutics Ltd.) (see,e.g., Clinical Trial Identifier: NCT02053220);

ONCOS-102 (previously called CGTG-102), which is an adenoviruscomprising granulocyte-macrophage colony stimulating factor (GM-CSF)(Oncos Therapeutics) (see, e.g., Clinical Trial Identifier:NCT01598129);

VCN-01, which is a genetically modified oncolytic human adenovirusencoding human PH20 hyaluronidase (VCN Biosciences, S.L.) (see, e.g.,Clinical Trial Identifiers: NCT02045602 and NCT02045589);

Conditionally Replicative Adenovirus ICOVIR-5, which is a virus derivedfrom wild-type human adenovirus serotype 5 (Had5) that has been modifiedto selectively replicate in cancer cells with a deregulatedretinoblastoma/E2F pathway (Institut Catalá d'Oncologia) (see, e.g.,Clinical Trial Identifier: NCT01864759);

Celyvir, which comprises bone marrow-derived autologous mesenchymal stemcells (MSCs) infected with ICOVIR5, an oncolytic adenovirus (HospitalInfantil Universitario Nifio Jesús, Madrid, Spain/Ramon Alemany) (see,e.g., Clinical Trial Identifier: NCT01844661);

CG0070, which is a conditionally replicating oncolytic serotype 5adenovirus (Ad5) in which human E2F-1 promoter drives expression of theessential E1a viral genes, thereby restricting viral replication andcytotoxicity to Rb pathway-defective tumor cells (Cold Genesys, Inc.)(see, e.g., Clinical Trial Identifier: NCT02143804); or

DNX-2401 (formerly named Delta-24-RGD), which is an adenovirus that hasbeen engineered to replicate selectively in retinoblastoma (Rb)-pathwaydeficient cells and to infect cells that express certain RGD-bindingintegrins more efficiently (Clinica Universidad de Navarra, Universidadde Navarra/DNAtrix, Inc.) (see, e.g., Clinical Trial Identifier:NCT01956734).

In some embodiments, an oncolytic virus described herein isadministering by injection, e.g., subcutaneous, intra-arterial,intravenous, intramuscular, intrathecal, or intraperitoneal injection.In embodiments, an oncolytic virus described herein is administeredintratumorally, transdermally, transmuco sally, orally, intranasally, orvia pulmonary administration.

In an embodiment, cells expressing a CAR described herein areadministered to a subject in combination with a molecule that decreasesthe Treg cell population. Methods that decrease the number of (e.g.,deplete) Treg cells are known in the art and include, e.g., CD25depletion, cyclophosphamide administration, modulating GITR function.Without wishing to be bound by theory, it is believed that reducing thenumber of Treg cells in a subject prior to apheresis or prior toadministration of a CAR-expressing cell described herein reduces thenumber of unwanted immune cells (e.g., Tregs) in the tumormicroenvironment and reduces the subject's risk of relapse.

In one embodiment, a CAR expressing cell described herein areadministered to a subject in combination with a molecule targeting GITRand/or modulating GITR functions, such as a GITR agonist and/or a GITRantibody that depletes regulatory T cells (Tregs). In embodiments, cellsexpressing a CAR described herein are administered to a subject incombination with cyclophosphamide. In one embodiment, the GITR bindingmolecules and/or molecules modulating GITR functions (e.g., GITR agonistand/or Treg depleting GITR antibodies) are administered prior toadministration of the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells. In embodiments, cyclophosphamide is administered to thesubject prior to administration (e.g., infusion or re-infusion) of theCAR-expressing cell or prior to apheresis of the cells. In embodiments,cyclophosphamide and an anti-GITR antibody are administered to thesubject prior to administration (e.g., infusion or re-infusion) of theCAR-expressing cell or prior to apheresis of the cells. In oneembodiment, the subject has cancer (e.g., a solid cancer or ahematological cancer such as ALL or CLL). In an embodiment, the subjecthas CLL. In embodiments, the subject has ALL. In embodiments, thesubject has a solid cancer, e.g., a solid cancer described herein.Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090,European Patent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT PublicationNos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibodydescribed, e.g., in U.S. Pat. No. 7,025,962, European Patent No.:1947183B1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, EuropeanPatent No.: EP 1866339, PCT Publication No.: WO 2011/028683, PCTPublication No.: WO 2013/039954, PCT Publication No.: WO2005/007190, PCTPublication No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCTPublication No.: WO 99/40196, PCT Publication No.: WO 2001/03720, PCTPublication No.: WO99/20758, PCT Publication No.: WO2006/083289, PCTPublication No.: WO 2005/115451, U.S. Pat. No. 7,618,632, and PCTPublication No.: WO 2011/051726.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a GITR agonist, e.g., aGITR agonist described herein. In one embodiment, the GITR agonist isadministered prior to the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with an mTOR inhibitor, e.g.,an mTOR inhibitor described herein, e.g., a rapalog such as everolimus.In one embodiment, the mTOR inhibitor is administered prior to theCAR-expressing cell. For example, in one embodiment, the mTOR inhibitorcan be administered prior to apheresis of the cells.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a protein tyrosinephosphatase inhibitor, e.g., a protein tyrosine phosphatase inhibitordescribed herein. In one embodiment, the protein tyrosine phosphataseinhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor describedherein, such as, e.g., sodium stibogluconate. In one embodiment, theprotein tyrosine phosphatase inhibitor is an SHP-2 inhibitor.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a kinase inhibitor. In one embodiment, the kinaseinhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein,e.g., a CD4/6 inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In one embodiment,the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitordescribed herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor describedherein. In one embodiment, the kinase inhibitor is a MNK inhibitor,e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine. The MNK inhibitorcan be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor. In oneembodiment, the kinase inhibitor is a DGK inhibitor, e.g., a DGKinhibitor described herein, such as, e.g., DGKinh1 (D5919) or DGKinh2(D5794). In one embodiment, the kinase inhibitor is a CDK4 inhibitorselected from aloisine A; flavopiridol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032);4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoicacid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g.,palbociclib (PD0332991), and the palbociclib is administered at a doseof about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125mg) daily for a period of time, e.g., daily for 14-21 days of a 28 daycycle, or daily for 7-12 days of a 21 day cycle. In one embodiment, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib areadministered.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a cyclin-dependent kinase (CDK) 4 or 6inhibitor, e.g., a CDK4 inhibitor or a CDK6 inhibitor described herein.In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a CDK4/6 inhibitor (e.g., an inhibitorthat targets both CDK4 and CDK6), e.g., a CDK4/6 inhibitor describedherein. In an embodiment, the subject has MCL. MCL is an aggressivecancer that is poorly responsive to currently available therapies, i.e.,essentially incurable. In many cases of MCL, cyclin D1 (a regulator ofCDK4/6) is expressed (e.g., due to chromosomal translocation involvingimmunoglobulin and Cyclin D1 genes) in MCL cells. Thus, without beingbound by theory, it is thought that MCL cells are highly sensitive toCDK4/6 inhibition with high specificity (i.e., minimal effect on normalimmune cells). CDK4/6 inhibitors alone have had some efficacy intreating MCL, but have only achieved partial remission with a highrelapse rate. An exemplary CDK4/6 inhibitor is LEE011 (also calledribociclib), the structure of which is shown below.

Without being bound by theory, it is believed that administration of aCAR-expressing cell described herein with a CDK4/6 inhibitor (e.g.,LEE011 or other CDK4/6 inhibitor described herein) can achieve higherresponsiveness, e.g., with higher remission rates and/or lower relapserates, e.g., compared to a CDK4/6 inhibitor alone.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected fromibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224;CC-292; ONO-4059; CNX-774; and LFM-A13. In a preferred embodiment, theBTK inhibitor does not reduce or inhibit the kinase activity ofinterleukin-2-inducible kinase (ITK), and is selected from GDC-0834;RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; andLFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g.,ibrutinib (PCI-32765). In embodiments, a CAR-expressing cell describedherein is administered to a subject in combination with a BTK inhibitor(e.g., ibrutinib). In embodiments, a CAR-expressing cell describedherein is administered to a subject in combination with ibrutinib (alsocalled PCI-32765). The structure of ibrutinib(1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one)is shown below.

In embodiments, the subject has CLL, mantle cell lymphoma (MCL), orsmall lymphocytic lymphoma (SLL). For example, the subject has adeletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject has relapsed CLL or SLL, e.g., the subjecthas previously been administered a cancer therapy (e.g., previously beenadministered one, two, three, or four prior cancer therapies). Inembodiments, the subject has refractory CLL or SLL. In otherembodiments, the subject has follicular lymphoma, e.g., relapse orrefractory follicular lymphoma. In some embodiments, ibrutinib isadministered at a dosage of about 300-600 mg/day (e.g., about 300-350,350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, e.g., about 420mg/day or about 560 mg/day), e.g., orally. In embodiments, the ibrutinibis administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g.,daily for 21 day cycle, or daily for 28 day cycle. In one embodiment, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib areadministered. In some embodiments, ibrutinib is administered incombination with rituximab. See, e.g., Burger et al. (2013) Ibrutinib InCombination With Rituximab (iR) Is Well Tolerated and Induces a HighRate Of Durable Remissions In Patients With High-Risk ChronicLymphocytic Leukemia (CLL): New, Updated Results Of a Phase II Trial In40 Patients, Abstract 675 presented at 55^(th) ASH Annual Meeting andExposition, New Orleans, La. 7-10 December. Without being bound bytheory, it is thought that the addition of ibrutinib enhances the T cellproliferative response and may shift T cells from a T-helper-2 (Th2) toT-helper-1 (Th1) phenotype. Th1 and Th2 are phenotypes of helper Tcells, with Th1 versus Th2 directing different immune response pathways.A Th1 phenotype is associated with proinflammatory responses, e.g., forkilling cells, such as intracellular pathogens/viruses or cancerouscells, or perpetuating autoimmune responses. A Th2 phenotype isassociated with eosinophil accumulation and anti-inflammatory responses.

In some embodiments of the methods, uses, and compositions herein, theBTK inhibitor is a BTK inhibitor described in International ApplicationWO/2015/079417, which is herein incorporated by reference in itsentirety. For instance, in some embodiments, the BTK inhibitor is acompound of formula (I) or a pharmaceutically acceptable salt thereof;

wherein,

R1 is hydrogen, C1-C6 alkyl optionally substituted by hydroxy;

R2 is hydrogen or halogen;

R3 is hydrogen or halogen;

R4 is hydrogen;

R5 is hydrogen or halogen;

or R4 and R5 are attached to each other and stand for a bond, —CH2-,—CH2-CH2-, —CH═CH—, —CH═CH—CH2-; —CH2-CH═CH—; or —CH2-CH2-CH2-;

R6 and R7 stand independently from each other for H, C1-C6 alkyloptionally substituted by hydroxyl, C3-C6 cycloalkyl optionallysubstituted by halogen or hydroxy, or halogen;

R8, R9, R, R′, R10 and R11 independently from each other stand for H, orC1-C6 alkyl optionally substituted by C1-C6 alkoxy; or any two of R8,R9, R, R′, R10 and R11 together with the carbon atom to which they arebound may form a 3-6 membered saturated carbocyclic ring;

R12 is hydrogen or C1-C6 alkyl optionally substituted by halogen orC1-C6 alkoxy;

or R12 and any one of R8, R9, R, R′, R10 or R11 together with the atomsto which they are bound may form a 4, 5, 6 or 7 membered azacyclic ring,which ring may optionally be substituted by halogen, cyano, hydroxyl,C1-C6 alkyl or C1-C6 alkoxy;

n is 0 or 1; and

R13 is C2-C6 alkenyl optionally substituted by C1-C6 alkyl, C1-C6 alkoxyor N,N-di-C1-C6 alkyl amino; C2-C6 alkynyl optionally substituted byC1-C6 alkyl or C1-C6 alkoxy; or C2-C6 alkylenyl oxide optionallysubstituted by C1-C6 alkyl.

In some embodiments, the BTK inhibitor of Formula I is chosen from:N-(3-(5-((1-Acryloylazetidin-3-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(E)-N-(3-(6-Amino-5-((1-(but-2-enoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-((1-propioloylazetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-((1-(but-2-ynoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-((1-Acryloylpiperidin-4-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-methylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(E)-N-(3-(6-Amino-5-(2-(N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-methylpropiolamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(E)-N-(3-(6-Amino-5-(2-(4-methoxy-N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(2-((4-Amino-6-(3-(4-cyclopropyl-2-fluorobenzamido)-5-fluoro-2-methylphenyl)pyrimidin-5-yl)oxy)ethyl)-N-methyloxirane-2-carboxamide;N-(2-((4-Amino-6-(3-(6-cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methylacrylamide;N-(3-(5-(2-Acrylamidoethoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-ethylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-(2-fluoroethyl)acrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-((1-Acrylamidocyclopropyl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(5-(2-Acrylamidopropoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(6-Amino-5-(2-(but-2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(6-Amino-5-(2-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(3-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(6-Amino-5-((1-(but-2-ynoyl)pyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-2-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one;N-(2-((4-Amino-6-(3-(6-cyclopropyl-1-oxo-3,4-dihydroisoquinolin-2(1H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methylacrylamide;N-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;2-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one;N-(3-(5-(((2S,4S)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(((2S,4S)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-(((2S,4R)-1-Acryloyl-4-fluoropyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-fluoropyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-N-(3-(6-Amino-5-((1-propioloylazetidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-2-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one;(R)-N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(R)-N-(3-(5-((1-Acryloylpiperidin-3-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-(((2R,3S)-1-Acryloyl-3-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-(((2S,4R)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;orN-(3-(5-(((2S,4S)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide.

Unless otherwise provided, the chemical terms used above in describingthe BTK inhibitor of Formula I are used according to their meanings asset out in International Application WO/2015/079417, which is hereinincorporated by reference in its entirety.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selectedfrom temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669; everolimus(RAD001); rapamycin (AY22989); simapimod;(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-mmino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 706), inner salt (SF1126); and XL765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g.,rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a periodof time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofrapamycin are administered. In one embodiment, the kinase inhibitor isan mTOR inhibitor, e.g., everolimus and the everolimus is administeredat a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for aperiod of time, e.g., daily for 28 day cycle. In one embodiment, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus areadministered.

In one embodiment, the kinase inhibitor is an MNK inhibitor selectedfrom CGP052088;4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d]pyrimidine (CGP57380);cercosporamide; ETC-1780445-2; and4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a phosphoinositide 3-kinase (PI3K)inhibitor (e.g., a PI3K inhibitor described herein, e.g., idelalisib orduvelisib) and/or rituximab. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withidelalisib and rituximab. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withduvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101;Gilead) is a small molecule that blocks the delta isoform of PI3K. Thestructure of idelalisib(5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone)is shown below.

Duvelisib (also called IPI-145; Infinity Pharmaceuticals and Abbvie) isa small molecule that blocks PI3K-δ,γ. The structure of duvelisib(8-Chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone)is shown below.

In embodiments, the subject has CLL. In embodiments, the subject hasrelapsed CLL, e.g., the subject has previously been administered acancer therapy (e.g., previously been administered an anti-CD20 antibodyor previously been administered ibrutinib). For example, the subject hasa deletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject comprises a leukemic cell comprising amutation in the immunoglobulin heavy-chain variable-region (IgV_(H))gene. In other embodiments, the subject does not comprise a leukemiccell comprising a mutation in the immunoglobulin heavy-chainvariable-region (IgV_(H)) gene. In embodiments, the subject has adeletion in the long arm of chromosome 11 (del(11q)). In otherembodiments, the subject does not have a del(11q). In embodiments,idelalisib is administered at a dosage of about 100-400 mg (e.g.,100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300,325-350, 350-375, or 375-400 mg), e.g., BID. In embodiments, duvelisibis administered at a dosage of about 15-100 mg (e.g., about 15-25,25-50, 50-75, or 75-100 mg), e.g., twice a day. In embodiments,rituximab is administered at a dosage of about 350-550 mg/m² (e.g.,350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m²), e.g.,intravenously.

In one embodiment, the kinase inhibitor is a dual phosphatidylinositol3-kinase (PI3K) and mTOR inhibitor selected from2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF-04691502);N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea(PF-05212384, PKI-587);2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile(BEZ-235); apitolisib (GDC-0980, RG7422);2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide(GSK2126458);8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-oneMaleic acid (NVP-BGT226);3-[4-(4-Morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]phenol(PI-103);5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(VS-5584, SB2343); andN-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide(XL765).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with an anaplastic lymphoma kinase (ALK)inhibitor. Exemplary ALK kinases include but are not limited tocrizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai),brigatinib (also called AP26113; Ariad), entrectinib (Ignyta),PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, e.g., Clinical TrialIdentifier No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). Insome embodiments, the subject has a solid cancer, e.g., a solid cancerdescribed herein, e.g., lung cancer.

The chemical name of crizotinib is3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine.The chemical name of ceritinib is5-Chloro-N²-[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]-N⁴-[2-(isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine.The chemical name of alectinib is9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile.The chemical name of brigatinib is5-Chloro-N²-{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N⁴-[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine.The chemical name of entrectinib isN-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)amino)benzamide.The chemical name of PF-06463922 is(10R)-7-Amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile.The chemical structure of CEP-37440 is(S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide.The chemical name of X-396 is(R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazine-1-carbonyl)phenyl)pyridazine-3-carboxamide.

Drugs that inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In a furtheraspect, the cell compositions of the present invention may beadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, and/orantibodies such as OKT3 or CAMPATH. In one aspect, the cell compositionsof the present invention are administered following B-cell ablativetherapy such as agents that react with CD20, e.g., Rituxan. For example,in one embodiment, subjects may undergo standard treatment with highdose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with an indoleamine 2,3-dioxygenase (IDO)inhibitor. IDO is an enzyme that catalyzes the degradation of the aminoacid, L-tryptophan, to kynurenine. Many cancers overexpress IDO, e.g.,prostatic, colorectal, pancreatic, cervical, gastric, ovarian, head, andlung cancer. pDCs, macrophages, and dendritic cells (DCs) can expressIDO. Without being bound by theory, it is thought that a decrease inL-tryptophan (e.g., catalyzed by IDO) results in an immunosuppressivemilieu by inducing T-cell anergy and apoptosis. Thus, without beingbound by theory, it is thought that an IDO inhibitor can enhance theefficacy of a CAR-expressing cell described herein, e.g., by decreasingthe suppression or death of a CAR-expressing immune cell. Inembodiments, the subject has a solid tumor, e.g., a solid tumordescribed herein, e.g., prostatic, colorectal, pancreatic, cervical,gastric, ovarian, head, or lung cancer. Exemplary inhibitors of IDOinclude but are not limited to 1-methyl-tryptophan, indoximod (NewLinkGenetics) (see, e.g., Clinical Trial Identifier Nos. NCT01191216;NCT01792050), and INCB024360 (Incyte Corp.) (see, e.g., Clinical TrialIdentifier Nos. NCT01604889; NCT01685255)

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a modulator of myeloid-derivedsuppressor cells (MDSCs). MDSCs accumulate in the periphery and at thetumor site of many solid tumors. These cells suppress T cell responses,thereby hindering the efficacy of CAR-expressing cell therapy. Withoutbeing bound by theory, it is thought that administration of a MDSCmodulator enhances the efficacy of a CAR-expressing cell describedherein. In an embodiment, the subject has a solid tumor, e.g., a solidtumor described herein, e.g., glioblastoma. Exemplary modulators ofMDSCs include but are not limited to MCS 110 and BLZ945. MCS 110 is amonoclonal antibody (mAb) against macrophage colony-stimulating factor(M-CSF). See, e.g., Clinical Trial Identifier No. NCT00757757. BLZ945 isa small molecule inhibitor of colony stimulating factor 1 receptor(CSF1R). See, e.g., Pyonteck et al. Nat. Med. 19(2013):1264-72. Thestructure of BLZ945 is shown below.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with an agent that inhibits or reduces theactivity of immunosuppressive plasma cells. Immunosuppressive plasmacells have been shown to impede T cell-dependent immunogenicchemotherapy, such as oxaliplatin (Shalapour et al., Nature 2015,521:94-101). In an embodiment, immunosuppressive plasma cells canexpress one or more of IgA, interleukin (IL)-10, and PD-L1. In anembodiment, the agent is a CD19 CAR-expressing cell or a BCMACAR-expressing cell.

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a interleukin-15 (IL-15)polypeptide, a interleukin-15 receptor alpha (IL-15Ra) polypeptide, or acombination of both a IL-15 polypeptide and a IL-15Ra polypeptide e.g.,hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimericnon-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described in,e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598, U.S. 2009/0082299,U.S. 2012/0141413, and U.S. 2011/0081311, incorporated herein byreference. In embodiments, het-IL-15 is administered subcutaneously. Inembodiments, the subject has a cancer, e.g., solid cancer, e.g.,melanoma or colon cancer. In embodiments, the subject has a metastaticcancer.

In embodiments, a subject having a disease described herein, e.g., ahematological disorder, e.g., AML or MDS, is administered aCAR-expressing cell described herein in combination with an agent, e.g.,cytotoxic or chemotherapy agent, a biologic therapy (e.g., antibody,e.g., monoclonal antibody, or cellular therapy), or an inhibitor (e.g.,kinase inhibitor). In embodiments, the subject is administered aCAR-expressing cell described herein in combination with a cytotoxicagent, e.g., CPX-351 (Celator Pharmaceuticals), cytarabine,daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine(Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. CPX-351 is aliposomal formulation comprising cytarabine and daunorubicin at a 5:1molar ratio. In embodiments, the subject is administered aCAR-expressing cell described herein in combination with ahypomethylating agent, e.g., a DNA methyltransferase inhibitor, e.g.,azacitidine or decitabine. In embodiments, the subject is administered aCAR-expressing cell described herein in combination with a biologictherapy, e.g., an antibody or cellular therapy, e.g., 225Ac-lintuzumab(Actimab-A; Actinium Pharmaceuticals), IPH2102 (Innate Pharma/BristolMyers Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin(Mylotarg; Pfizer). SGN-CD33A is an antibody-drug conjugate (ADC)comprising a pyrrolobenzodiazepine dimer that is attached to ananti-CD33 antibody. Actimab-A is an anti-CD33 antibody (lintuzumab)labeled with actinium. IPH2102 is a monoclonal antibody that targetskiller immunoglobulin-like receptors (KIRs). In embodiments, the subjectis administered a CAR-expressing cell described herein in combination aFLT3 inhibitor, e.g., sorafenib (Bayer), midostaurin (Novartis),quizartinib (Daiichi Sankyo), crenolanib (Arog Pharmaceuticals), PLX3397(Daiichi Sankyo), AKN-028 (Akinion Pharmaceuticals), or ASP2215(Astellas). In embodiments, the subject is administered a CAR-expressingcell described herein in combination with an isocitrate dehydrogenase(IDH) inhibitor, e.g., AG-221 (Celgene/Agios) or AG-120 (Agios/Celgene).In embodiments, the subject is administered a CAR-expressing celldescribed herein in combination with a cell cycle regulator, e.g.,inhibitor of polo-like kinase 1 (Plk1), e.g., volasertib (BoehringerIngelheim); or an inhibitor of cyclin-dependent kinase 9 (Cdk9), e.g.,alvocidib (Tolero Pharmaceuticals/Sanofi Aventis). In embodiments, thesubject is administered a CAR-expressing cell described herein incombination with a B cell receptor signaling network inhibitor, e.g., aninhibitor of B-cell lymphoma 2 (Bcl-2), e.g., venetoclax (Abbvie/Roche);or an inhibitor of Bruton's tyrosine kinase (Btk), e.g., ibrutinib(Pharmacyclics/Johnson & Johnson Janssen Pharmaceutical). Inembodiments, the subject is administered a CAR-expressing cell describedherein in combination with an inhibitor of M1 aminopeptidase, e.g.,tosedostat (CTI BioPharma/Vernalis); an inhibitor of histone deacetylase(HDAC), e.g., pracinostat (MEI Pharma); a multi-kinase inhibitor, e.g.,rigosertib (Onconova Therapeutics/Baxter/SymBio); or a peptidic CXCR4inverse agonist, e.g., BL-8040 (BioLineRx). In embodiments, the subjectis administered a CD123-targeting CAR-expressing cell in combinationwith a CAR-expressing cell that targets an antigen other than CD123,e.g., CLL-1, BCMA, CD33, CD19, FLT-3, or folate receptor beta.

In another embodiment, the subjects receive an infusion of the CD123CAR-expressing cell compositions of the present invention prior totransplantation, e.g., allogeneic stem cell transplant, of cells. In apreferred embodiment, CD123-CAR expressing cells transiently expressCD123 CAR, e.g., by electroporation of an mRNA CD123 CAR, whereby theexpression of the CD123 is terminated prior to infusion of donor stemcells to avoid engraftment failure.

Some patients may experience allergic reactions to the compounds of thepresent invention and/or other anti-cancer agent(s) during or afteradministration; therefore, anti-allergic agents are often administeredto minimize the risk of an allergic reaction. Suitable anti-allergicagents include corticosteroids, such as dexamethasone (e.g., Decadron®),beclomethasone (e.g., Beclovent®), hydrocortisone (also known ascortisone, hydrocortisone sodium succinate, hydrocortisone sodiumphosphate, and sold under the tradenames Ala-Cort®, hydrocortisonephosphate, Solu-Cortef®, Hydrocort Acetate® and Lanacort®), prednisolone(sold under the tradenames Delta-Cortel®, Orapred®, Pediapred® andPrelone®), prednisone (sold under the tradenames Deltasone®, LiquidRed®, Meticorten® and Orasone®), methylprednisolone (also known as6-methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, sold under the tradenames Duralone®, Medralone®,Medrol®, M-Prednisol® and Solu-Medrol®); antihistamines, such asdiphenhydramine (e.g., Benadryl®), hydroxyzine, and cyproheptadine; andbronchodilators, such as the beta-adrenergic receptor agonists,albuterol (e.g., Proventil®), and terbutaline (Brethine®).

Some patients may experience nausea during and after administration ofthe compound of the present invention and/or other anti-cancer agent(s);therefore, anti-emetics are used in preventing nausea (upper stomach)and vomiting. Suitable anti-emetics include aprepitant (Emend®),ondansetron (Zofran®), granisetron HCl (Kytril®), lorazepam (Ativan®.dexamethasone (Decadron®), prochlorperazine (Compazine®), casopitant(Rezonic® and Zunrisa®), and combinations thereof.

Medication to alleviate the pain experienced during the treatment periodis often prescribed to make the patient more comfortable. Commonover-the-counter analgesics, such Tylenol®, are often used. However,opioid analgesic drugs such as hydrocodone/paracetamol orhydrocodone/acetaminophen (e.g., Vicodin®), morphine (e.g., Astramorph®or Avinza®), oxycodone (e.g., OxyContin® or Percocet®), oxymorphonehydrochloride (Opana®), and fentanyl (e.g., Duragesic®) are also usefulfor moderate or severe pain.

In an effort to protect normal cells from treatment toxicity and tolimit organ toxicities, cytoprotective agents (such as neuroprotectants,free-radical scavengers, cardioprotectors, anthracycline extravasationneutralizers, nutrients and the like) may be used as an adjunct therapy.Suitable cytoprotective agents include Amifostine (Ethyol®), glutamine,dimesna (Tavocept®), mesna (Mesnex®), dexrazoxane (Zinecard® orTotect®), xaliproden (Xaprila®), and leucovorin (also known as calciumleucovorin, citrovorum factor and folinic acid).

The structure of the active compounds identified by code numbers,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications).

The above-mentioned compounds, which can be used in combination with acompound of the present invention, can be prepared and administered asdescribed in the art, such as in the documents cited above.

In one embodiment, the present invention provides pharmaceuticalcompositions comprising at least one compound of the present invention(e.g., a compound of the present invention) or a pharmaceuticallyacceptable salt thereof together with a pharmaceutically acceptablecarrier suitable for administration to a human or animal subject, eitheralone or together with other anti-cancer agents.

In one embodiment, the present invention provides methods of treatinghuman or animal subjects suffering from a cellular proliferativedisease, such as cancer. The present invention provides methods oftreating a human or animal subject in need of such treatment, comprisingadministering to the subject a therapeutically effective amount of acompound of the present invention (e.g., a compound of the presentinvention) or a pharmaceutically acceptable salt thereof, either aloneor in combination with other anti-cancer agents.

In particular, compositions will either be formulated together as acombination therapeutic or administered separately.

In combination therapy, the compound of the present invention and otheranti-cancer agent(s) may be administered either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of the patient.

In a preferred embodiment, the compound of the present invention and theother anti-cancer agent(s) is generally administered sequentially in anyorder by infusion or orally. The dosing regimen may vary depending uponthe stage of the disease, physical fitness of the patient, safetyprofiles of the individual drugs, and tolerance of the individual drugs,as well as other criteria well-known to the attending physician andmedical practitioner(s) administering the combination. The compound ofthe present invention and other anti-cancer agent(s) may be administeredwithin minutes of each other, hours, days, or even weeks apart dependingupon the particular cycle being used for treatment. In addition, thecycle could include administration of one drug more often than the otherduring the treatment cycle and at different doses per administration ofthe drug.

In another aspect of the present invention, kits that include one ormore compound of the present invention and a combination partner asdisclosed herein are provided. Representative kits include (a) acompound of the present invention or a pharmaceutically acceptable saltthereof, (b) at least one combination partner, e.g., as indicated above,whereby such kit may comprise a package insert or other labelingincluding directions for administration.

A compound of the present invention may also be used to advantage incombination with known therapeutic processes, for example, theadministration of hormones or especially radiation. A compound of thepresent invention may in particular be used as a radiosensitizer,especially for the treatment of tumors which exhibit poor sensitivity toradiotherapy.

In one embodiment, the subject can be administered an agent whichreduces or ameliorates a side effect associated with the administrationof a CAR-expressing cell. Side effects associated with theadministration of a CAR-expressing cell include, but are not limited toCRS, and hemophagocytic lymphohistiocytosis (HLH), also termedMacrophage Activation Syndrome (MAS). Symptoms of CRS include highfevers, nausea, transient hypotension, hypoxia, and the like. CRS mayinclude clinical constitutional signs and symptoms such as fever,fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, and headache.CRS may include clinical skin signs and symptoms such as rash. CRS mayinclude clinical gastrointestinal signs and symptoms such as nausea,vomiting and diarrhea. CRS may include clinical respiratory signs andsymptoms such as tachypnea and hypoxemia. CRS may include clinicalcardiovascular signs and symptoms such as tachycardia, widened pulsepressure, hypotension, increased cardiac output (early) and potentiallydiminished cardiac output (late). CRS may include clinical coagulationsigns and symptoms such as elevated d-dimer, hypofibrinogenemia with orwithout bleeding. CRS may include clinical renal signs and symptoms suchas azotemia. CRS may include clinical hepatic signs and symptoms such astransaminitis and hyperbilirubinemia. CRS may include clinicalneurologic signs and symptoms such as headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dymetria, altered gait, and seizures.

Accordingly, the methods described herein can comprise administering aCAR-expressing cell described herein to a subject and furtheradministering one or more agents to manage elevated levels of a solublefactor resulting from treatment with a CAR-expressing cell. In oneembodiment, the soluble factor elevated in the subject is one or more ofIFN-γ, TNFα, IL-2 and IL-6. In an embodiment, the factor elevated in thesubject is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5 andfraktalkine. Therefore, an agent administered to treat this side effectcan be an agent that neutralizes one or more of these soluble factors.In one embodiment, the agent that neutralizes one or more of thesesoluble forms is an antibody or antigen binding fragment thereof.Examples of such agents include, but are not limited to a steroid (e.g.,corticosteroid), an inhibitor of TNFα, and an inhibitor of IL-6. Anexample of a TNFα inhibitor is an anti-TNFα antibody molecule such as,infliximab, adalimumab, certolizumab pegol, and golimumab. Anotherexample of a TNFα inhibitor is a fusion protein such as entanercept.Small molecule inhibitor of TNFα include, but are not limited to,xanthine derivatives (e.g. pentoxifylline) and bupropion. An example ofan IL-6 inhibitor is an anti-IL-6 antibody molecule such as tocilizumab(toc), sarilumab, elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136,CPSI-2364, CDP6038, VX30, ARGX-109, FE301, and FM101. In one embodiment,the anti-IL-6 antibody molecule is tocilizumab. An example of an IL-1Rbased inhibitor is anakinra.

In some embodiment, the subject is administered a corticosteroid, suchas, e.g., methylprednisolone, hydrocortisone, among others.

In some embodiments, the subject is administered a vasopressor, such as,e.g., norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin,or a combination thereof.

In an embodiment, the subject can be administered an antipyretic agent.In an embodiment, the subject can be administered an analgesic agent.

In one embodiment, the subject can be administered an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule, e.g., the agent is a checkpoint inhibitor. Inhibitorymolecules, e.g., Programmed Death 1 (PD1), can, in some embodiments,decrease the ability of a CAR-expressing cell to mount an immuneeffector response. Examples of inhibitory molecules include PD1, PD-L1,CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA,RNA or protein level, can optimize a CAR-expressing cell performance. Inembodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularlyinterspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used to inhibitexpression of an inhibitory molecule in the CAR-expressing cell. In anembodiment the inhibitor is an shRNA. In an embodiment, the inhibitorymolecule is inhibited within a CAR-expressing cell. In theseembodiments, a dsRNA molecule that inhibits expression of the inhibitorymolecule is linked to the nucleic acid that encodes a component, e.g.,all of the components, of the CAR.

In an embodiment, a nucleic acid molecule that encodes a dsRNA moleculethat inhibits expression of the molecule that modulates or regulates,e.g., inhibits, T-cell function is operably linked to a promoter, e.g.,a H1- or a U6-derived promoter such that the dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is expressed, e.g., is expressed within aCAR-expressing cell. See e.g., Tiscornia G., “Development of LentiviralVectors Expressing siRNA,” Chapter 3, in Gene Transfer: Delivery andExpression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA, 2007; Brummelkamp T R,et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat.Biotechnol. 19: 497-500. In an embodiment the nucleic acid molecule thatencodes a dsRNA molecule that inhibits expression of the molecule thatmodulates or regulates, e.g., inhibits, T-cell function is present onthe same vector, e.g., a lentiviral vector, that comprises a nucleicacid molecule that encodes a component, e.g., all of the components, ofthe CAR. In such an embodiment, the nucleic acid molecule that encodes adsRNA molecule that inhibits expression of the molecule that modulatesor regulates, e.g., inhibits, T-cell function is located on the vector,e.g., the lentiviral vector, 5′- or 3′- to the nucleic acid that encodesa component, e.g., all of the components, of the CAR. The nucleic acidmolecule that encodes a dsRNA molecule that inhibits expression of themolecule that modulates or regulates, e.g., inhibits, T-cell functioncan be transcribed in the same or different direction as the nucleicacid that encodes a component, e.g., all of the components, of the CAR.

In an embodiment the nucleic acid molecule that encodes a dsRNA moleculethat inhibits expression of the molecule that modulates or regulates,e.g., inhibits, T-cell function is present on a vector other than thevector that comprises a nucleic acid molecule that encodes a component,e.g., all of the components, of the CAR. In an embodiment, the nucleicacid molecule that encodes a dsRNA molecule that inhibits expression ofthe molecule that modulates or regulates, e.g., inhibits, T-cellfunction it transiently expressed within a CAR-expressing cell. In anembodiment, the nucleic acid molecule that encodes a dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is stably integrated into the genome of aCAR-expressing cell. FIGS. 51A-51E depicts examples of vectors forexpressing a component, e.g., all of the components, of the CAR with adsRNA molecule that inhibits expression of the molecule that modulatesor regulates, e.g., inhibits, T-cell function.

Examples of dsRNA molecules useful for inhibiting expression of amolecule that modulates or regulates, e.g., inhibits, T-cell function,wherein the molecule that modulates or regulates, e.g., inhibits, T-cellfunction is PD-1 are provided below.

Provided in Table 16 below are the names of PDCD1 (PD1) RNAi agents(derived from their position in the mouse PDCD1 gene sequenceNM_008798.2), along with the SEQ ID NOs: 216-263 representing the DNAsequence. Both sense (S) and antisense (AS) sequences are presented as19mer and 21mer sequences are in this table. Also note that the position(PoS, e.g., 176) is derived from the position number in the mouse PDCD1gene sequence NM_008798.2. SEQ ID NOs are indicated in groups of 12 thatcorrespond with “sense 19” SEQ ID NOs: 608-619; “sense 21” SEQ ID NOs:620-631; “asense 21” SEQ ID NOs: 632-643; “asense 19” SEQ ID NOs:644-655.

TABLE 16  Mouse PDCD1 (PD1) shRNA sequences Position on TargetNM_008798.2 region Sense19 Sense21 Asense21 Asense19  176 CDSGGAGGTCCCTC CTGGAGGTCCC TAGAAGGTGAG TAGAAGGTGAG ACCTTCTA  TCACCTTCTAGGACCTCCAG GGACCTCC  (SEQ ID NO: 608) (SEQ ID NO: 620) (SEQ ID NO: 632)(SEQ ID NO: 644)  260 CDS CGGAGGATCTT GTCGGAGGATC TTCAGCATAAGTTCAGCATAAG ATGCTGAA TTATGCTGAA ATCCTCCGAC ATCCTCCG (SEQ ID NO: 609)(SEQ ID NO: 621) (SEQ ID NO: 633) (SEQ ID NO: 645)  359 CDS CCCGCTTCCAGTGCCCGCTTCC TGTATGATCTG TGTATGATCTG ATCATACA AGATCATACA GAAGCGGGCAGAAGCGGG (SEQ ID NO: 610) (SEQ ID NO: 622) (SEQ ID NO: 634)(SEQ ID NO: 646)  528 CDS GGAGACCTCAA CTGGAGACCTC ATATCTTGTTGAATATCTTGTTG CAAGATAT AACAAGATAT GGTCTCCAG AGGTCTCC (SEQ ID NO: 611)(SEQ ID NO: 623) (SEQ ID NO: 635) (SEQ ID NO: 647)  581 CDS AAGGCATGGTCTCAAGGCATGG ATACCAATGAC ATACCAATGAC ATTGGTAT TCATTGGTAT CATGCCTTGACATGCCTT (SEQ ID NO: 612) (SEQ ID NO: 624) (SEQ ID NO: 636)(SEQ ID NO: 648)  584 CDS GCATGGTCATT AGGCATGGTCA ATGATACCAATATGATACCAAT GGTATCAT (SEQ TTGGTATCAT GACCATGCCT GACCATGC ID NO: 613)(SEQ ID NO: 625) (SEQ ID NO: 637) (SEQ ID NO: 649)  588 CDS GGTCATTGGTAATGGTCATTGG ATGGTCATTGG ATGGTCATTGG TCATGAGT (SEQ TATCATGAGT TATCATGAGTTATCATGA (SEQ ID NO: 614) (SEQ ID NO: 626) (SEQ ID NO: 638) ID NO: 650) 609 CDS CCTAGTGGGTA GCCCTAGTGGG GCCCTAGTGGG GCCCTAGTGGG TCCCTGTA (SEQTATCCCTGTA TATCCCTGTA TATCCCTG ID NO: 615) (SEQ ID NO: 627)(SEQ ID NO: 639) (SEQ ID NO: 651)  919 CDS GAGGATGGACA ATGAGGATGGAATGAGGATGGA ATGAGGATGGA TTGTTCTT CATTGTTCTT CATTGTTCTT CATTGTTC(SEQ ID NO: 616) (SEQ ID NO: 628) (SEQ ID NO: 640) (SEQ ID NO: 652) 10213′UTR GCATGCAGGCT GAGCATGCAGG GAGCATGCAGG GAGCATGCAGG ACAGTTCA (SEQCTACAGTTCA CTACAGTTCA CTACAGTT ID NO: 617) (SEQ ID NO: 629)(SEQ ID NO: 641) (SEQ ID NO: 653) 1097 3′UTR CCAGCACATGC TTCCAGCACATTTCCAGCACAT TTCCAGCACAT ACTGTTGA (SEQ GCACTGTTGA GCACTGTTGA GCACTGTTID NO: 618) (SEQ ID NO: 630) (SEQ ID NO: 642) (SEQ ID NO: 654) 11013′UTR CACATGCACTG AGCACATGCAC AGCACATGCAC AGCACATGCAC TTGAGTGA (SEQTGTTGAGTGA TGTTGAGTGA TGTTGAGT ID NO: 619) (SEQ ID NO: 631)(SEQ ID NO: 643) (SEQ ID NO: 655)

Provided in Table 17 below are the names of PDCD1 (PD1) RNAi agents(derived from their position in the human PDCD1 gene sequence, alongwith the SEQ ID NOs. 264-311 representing the DNA sequence. Both sense(S) and antisense (AS) sequences are presented as 19mer and 21mersequences. SEQ ID NOs are indicated in groups of 12 that correspond with“sense 19” SEQ ID NOs: 656-667; “sense 21” SEQ ID NOs: 668-679; “asense21” SEQ ID NOs: 680-691; “asense 19” SEQ ID NOs: 692-703.

TABLE 17  Human PDCD1 (PD1) shRNA sequences Position on TargetNM_005018.2 region Sense19 Asense19 Sense21 Asense21 145 CDS GGCCAGGATGGTCTAAGAACCA GCGGCCAGGAT TCTAAGAACCA TTCTTAGA (SEQ TCCTGGCC (SEQGGTTCTTAGA  TCCTGGCCGC ID NO: 656) ID NO: 668) (SEQ ID NO: (SEQ ID NO:680) 692) 271 CDS GCTTCGTGCTA TACCAGTTTAG GAGCTTCGTGC TACCAGTTTAGAACTGGTA (SEQ CACGAAGC (SEQ TAAACTGGTA CACGAAGCTC ID NO: 657)ID NO: 669) (SEQ ID NO: (SEQ ID NO: 681) 693) 393 CDS GGGCGTGACTTTCATGTGGAAG ACGGGCGTGAC TCATGTGGAAG CCACATGA (SEQ TCACGCCC (SEQTTCCACATGA TCACGCCCGT ID NO: 658) ID NO: 670) (SEQ ID NO: (SEQ ID NO:682) 694) 1497 3′UTR CAGGCCTAGAG TGAAACTTCTC TGCAGGCCTAG TGAAACTTCTCAAGTTTCA (SEQ TAGGCCTG (SEQ AGAAGTTTCA TAGGCCTGCA ID NO: 659)ID NO: 671) (SEQ ID NO: (SEQ ID NO: 683) 695) 1863 3′UTR CTTGGAACCCATTCAGGAATGG TCCTTGGAACC TTCAGGAATGG TTCCTGAA (SEQ GTTCCAAG (SEQCATTCCTGAA GTTCCAAGGA ID NO: 660) ID NO: 672) (SEQ ID NO: (SEQ ID NO:684) 696) 1866 3′UTR GGAACCCATTC AATTTCAGGAA TTGGAACCCAT AATTTCAGGAACTGAAATT (SEQ TGGGTTCC (SEQ TCCTGAAATT TGGGTTCCAA ID NO: 661)ID NO: 673) (SEQ ID NO: (SEQ ID NO: 685) 697) 1867 3′UTR GAACCCATTCCTAATTTCAGGA TGGAACCCATT TAATTTCAGGA TGAAATTA (SEQ ATGGGTTC (SEQCCTGAAATTA ATGGGTTCCA ID NO: 662) ID NO: 674) (SEQ ID NO: (SEQ ID NO:686) 698) 1868 3′UTR AACCCATTCCT ATAATTTCAGG GGAACCCATTC ATAATTTCAGGGAAATTAT (SEQ AATGGGTT (SEQ CTGAAATTAT AATGGGTTCC ID NO: 663)ID NO: 675) (SEQ ID NO: (SEQ ID NO: 687) 699) 1869 3′UTR ACCCATTCCTGAATAATTTCAG GAACCCATTCC AATAATTTCAG AAATTATT (SEQ GAATGGGT (SEQTGAAATTATT GAATGGGTTC ID NO: 664) ID NO: 676) (SEQ ID NO: (SEQ ID NO:688) 700) 1870 3′UTR CCCATTCCTGA AAATAATTTCA AACCCATTCCT AAATAATTTCAAATTATTT (SEQ GGAATGGG (SEQ GAAATTATTT GGAATGGGTT ID NO: 665)ID NO: 677) (SEQ ID NO: (SEQ ID NO: 689) 701) 2079 3′UTR CTGTGGTTCTATTAATATAATAG CCCTGTGGTTCT TAATATAATAG TATATTA (SEQ AACCACAG (SEQATTATATTA AACCACAGGG ID NO: 666) ID NO: 678) (SEQ ID NO: (SEQ ID NO:690) 702) 2109 3′UTR AAATATGAGAG TTAGCATGCTC TTAAATATGAG TTAGCATGCTCCATGCTAA (SEQ TCATATTT (SEQ AGCATGCTAA TCATATTTAA ID NO: 667)ID NO: 679) (SEQ ID NO: (SEQ ID NO:  691) 703)

In one embodiment, the inhibitor of an inhibitory signal can be, e.g.,an antibody or antibody fragment that binds to an inhibitory molecule.For example, the agent can be an antibody or antibody fragment thatbinds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred toas MDX-010 and MDX-101, and marketed as Yervoy®; Bristol-Myers Squibb;Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerlyknown as ticilimumab, CP-675,206).). In an embodiment, the agent is anantibody or antibody fragment that binds to TIM3. In an embodiment, theagent is an antibody or antibody fragment that binds to LAG3. Inembodiments, the agent that enhances the activity of a CAR-expressingcell, e.g., inhibitor of an inhibitory molecule, is administered incombination with an allogeneic CAR, e.g., an allogeneic CAR describedherein (e.g., described in the Allogeneic CAR section herein).

PD-1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated Bcells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol8:765-75). Two ligands for PD-1, PD-L1 and PD-L2 have been shown todownregulate T cell activation upon binding to PD-1 (Freeman et a. 2000J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carteret al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers(Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol.Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094).Immune suppression can be reversed by inhibiting the local interactionof PD-1 with PD-L1.

Antibodies, antibody fragments, and other inhibitors of PD-1, PD-L1 andPD-L2 are available in the art and may be used combination with a carsof the present invention described herein. For example, nivolumab (alsoreferred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fullyhuman IgG4 monoclonal antibody which specifically blocks PD-1. Nivolumab(clone 5C4) and other human monoclonal antibodies that specifically bindto PD-1 are disclosed in U.S. Pat. No. 8,008,449 and WO2006/121168.Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibodythat binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonalantibodies are disclosed in WO2009/101611. Pembrolizumab (formerly knownas lambrolizumab, and also referred to as MK03475; Merck) is a humanizedIgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and otherhumanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibodythat binds to PDL1, and inhibits interaction of the ligand with PD1.MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonalantibody that binds to PD-L1. MDPL3280A and other human monoclonalantibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.SPublication No.: 20120039906. Other anti-PD-L1 binding agents includeYW243.55.570 (heavy and light chain variable regions are shown in SEQ IDNOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referred to asBMS-936559, and, e.g., anti-PD-L1 binding agents disclosed inWO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed inWO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptorthat blocks the interaction between PD-1 and B7-H1. Other anti-PD-1antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/orUS 20120114649.

In one embodiment, the anti-PD-1 antibody or fragment thereof is ananti-PD-1 antibody molecule as described in US 2015/0210769, entitled“Antibody Molecules to PD-1 and Uses Thereof,” incorporated by referencein its entirety. In one embodiment, the anti-PD-1 antibody moleculeincludes at least one, two, three, four, five or six CDRs (orcollectively all of the CDRs) from a heavy and light chain variableregion from an antibody chosen from any of BAP049-hum01, BAP049-hum02,BAP049-hum03, BAP049-hum04, BAP049-hum05, BAP049-hum06, BAP049-hum07,BAP049-hum08, BAP049-hum09, BAP049-hum10, BAP049-hum11, BAP049-hum12,BAP049-hum13, BAP049-hum14, BAP049-hum15, BAP049-hum16, BAP049-Clone-A,BAP049-Clone-B, BAP049-Clone-C, BAP049-Clone-D, or BAP049-Clone-E; or asdescribed in Table 1 of US 2015/0210769, or encoded by the nucleotidesequence in Table 1, or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to anyof the aforesaid sequences; or closely related CDRs, e.g., CDRs whichare identical or which have at least one amino acid alteration, but notmore than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions).

In yet another embodiment, the anti-PD-1 antibody molecule comprises atleast one, two, three or four variable regions from an antibodydescribed herein, e.g., an antibody chosen from any of BAP049-hum01,BAP049-hum02, BAP049-hum03, BAP049-hum04, BAP049-hum05, BAP049-hum06,BAP049-hum07, BAP049-hum08, BAP049-hum09, BAP049-hum10, BAP049-hum11,BAP049-hum12, BAP049-hum13, BAP049-hum14, BAP049-hum15, BAP049-hum16,BAP049-Clone-A, BAP049-Clone-B, BAP049-Clone-C, BAP049-Clone-D, orBAP049-Clone-E; or as described in Table 1 of US 2015/0210769, orencoded by the nucleotide sequence in Table 1; or a sequencesubstantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical) to any of the aforesaid sequences.

TIM3 (T cell immunoglobulin-3) also negatively regulates T cellfunction, particularly in IFN-g-secreting CD4+ T helper 1 and CD8+ Tcytotoxic 1 cells, and plays a critical role in T cell exhaustion.Inhibition of the interaction between TIM3 and its ligands, e.g.,galectin-9 (Ga19), phosphotidylserine (PS), and HMGB1, can increaseimmune response. Antibodies, antibody fragments, and other inhibitors ofTIM3 and its ligands are available in the art and may be usedcombination with a CD19 CAR described herein. For example, antibodies,antibody fragments, small molecules, or peptide inhibitors that targetTIM3 binds to the IgV domain of TIM3 to inhibit interaction with itsligands. Antibodies and peptides that inhibit TIM3 are disclosed inWO2013/006490 and US20100247521. Other anti-TIM3 antibodies includehumanized versions of RMT3-23 (disclosed in Ngiow et al., 2011, CancerRes, 71:3540-3551), and clone 8B.2C12 (disclosed in Monney et al., 2002,Nature, 415:536-541). Bi-specific antibodies that inhibit TIM3 and PD-1are disclosed in US20130156774.

In one embodiment, the anti-TIM3 antibody or fragment thereof is ananti-TIM3 antibody molecule as described in US 2015/0218274, entitled“Antibody Molecules to TIM3 and Uses Thereof,” incorporated by referencein its entirety. In one embodiment, the anti-TIM3 antibody moleculeincludes at least one, two, three, four, five or six CDRs (orcollectively all of the CDRs) from a heavy and light chain variableregion from an antibody chosen from any of ABTIM3, ABTIM3-hum01,ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05, ABTIM3-hum06,ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum11,ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15, ABTIM3-hum16,ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20, ABTIM3-hum21,ABTIM3-hum22, ABTIM3-hum23; or as described in Tables 1-4 of US2015/0218274; or encoded by the nucleotide sequence in Tables 1-4; or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences, or closely related CDRs, e.g., CDRs which are identical orwhich have at least one amino acid alteration, but not more than two,three or four alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions).

In yet another embodiment, the anti-TIM3 antibody molecule comprises atleast one, two, three or four variable regions from an antibodydescribed herein, e.g., an antibody chosen from any of ABTIM3,ABTIM3-hum01, ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05,ABTIM3-hum06, ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10,ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15,ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20,ABTIM3-hum21, ABTIM3-hum22, ABTIM3-hum23; or as described in Tables 1-4of US 2015/0218274; or encoded by the nucleotide sequence in Tables 1-4;or a sequence substantially identical (e.g., at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences

In other embodiments, the agent which enhances the activity of aCAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3,and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAMis an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodiesare described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or arecombinant form thereof, as described in, e.g., US 2004/0047858, U.S.Pat. No. 7,132,255 and WO 99/052552. In other embodiments, theanti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng etal. PLoS One. 2010 Sep. 2; 5(9). pii: e12529 (DOI:10:1371/journal.pone.0021146), or crossreacts with CEACAM-1 and CEACAM-5as described in, e.g., WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, carcinoembryonic antigen celladhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, are believedto mediate, at least in part, inhibition of an anti-tumor immuneresponse (see e.g., Markel et al. J Immunol. 2002 Mar. 15;168(6):2803-10; Markel et al. J Immunol. 2006 Nov. 1; 177(9):6062-71;Markel et al. Immunology. 2009 February; 126(2):186-200; Markel et al.Cancer Immunol Immunother. 2010 February; 59(2):215-30; Ortenberg et al.Mol Cancer Ther. 2012 June; 11(6):1300-10; Stern et al. J Immunol. 2005Jun. 1; 174(11):6692-701; Zheng et al. PLoS One. 2010 Sep. 2; 5(9). pii:e12529). For example, CEACAM-1 has been described as a heterophilicligand for TIM-3 and as playing a role in TIM-3-mediated T celltolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al. (2014)Nature doi:10.1038/nature13848). In embodiments, co-blockade of CEACAM-1and TIM-3 has been shown to enhance an anti-tumor immune response inxenograft colorectal cancer models (see e.g., WO 2014/022332; Huang, etal. (2014), supra). In other embodiments, co-blockade of CEACAM-1 andPD-1 reduce T cell tolerance as described, e.g., in WO 2014/059251.Thus, CEACAM inhibitors can be used with the other immunomodulatorsdescribed herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) toenhance an immune response against a cancer, e.g., a melanoma, a lungcancer (e.g., NSCLC), a bladder cancer, a colon cancer an ovariancancer, and other cancers as described herein.

LAG3 (lymphocyte activation gene-3 or CD223) is a cell surface moleculeexpressed on activated T cells and B cells that has been shown to play arole in CD8+ T cell exhaustion. Antibodies, antibody fragments, andother inhibitors of LAG3 and its ligands are available in the art andmay be used combination with a CD19 CAR described herein. For example,BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targetsLAG3. IMP701 (Imrnutep) is an antagonist LAG3 antibody and IMP731(Immutep and GlaxoSmithKline) is depleting LAG3 antibody. Other LAG3inhibitors include IMP321 (Immutep), which is a recombinant fusionprotein of a soluble portion of LAG3 and Ig that binds to WIC class iimolecules and activates antigen presenting cells (APC). Other antibodiesare disclosed, e.g., in WO2010/019570.

In some embodiments, the agent which enhances the activity of aCAR-expressing cell can be, e.g., a fusion protein comprising a firstdomain and a second domain, wherein the first domain is an inhibitorymolecule, or fragment thereof, and the second domain is a polypeptidethat is associated with a positive signal, e.g., a polypeptidecomprising an antracellular signaling domain as described herein. Insome embodiments, the polypeptide that is associated with a positivesignal can include a costimulatory domain of CD28, CD27, ICOS, e.g., anintracellular signaling domain of CD28, CD27 and/or ICOS, and/or aprimary signaling domain, e.g., of CD3 zeta, e.g., described herein. Inone embodiment, the fusion protein is expressed by the same cell thatexpressed the CAR. In another embodiment, the fusion protein isexpressed by a cell, e.g., a T cell that does not express a CD123 CAR.

In one embodiment, the agent which enhances activity of a CAR-expressingcell described herein is miR-17-92.

In one embodiment, the agent which enhances activity of a CAR-describedherein is a cytokine. Cytokines have important functions related to Tcell expansion, differentiation, survival, and homeostatis. Cytokinesthat can be administered to the subject receiving a CAR-expressing celldescribed herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, andIL-21, or a combination thereof. In preferred embodiments, the cytokineadministered is IL-7, IL-15, or IL-21, or a combination thereof. Thecytokine can be administered once a day or more than once a day, e.g.,twice a day, three times a day, or four times a day. The cytokine can beadministered for more than one day, e.g. the cytokine is administeredfor 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or4 weeks. For example, the cytokine is administered once a day for 7days.

In embodiments, the cytokine is administered in combination withCAR-expressing T cells. The cytokine can be administered simultaneouslyor concurrently with the CAR-expressing T cells, e.g., administered onthe same day. The cytokine may be prepared in the same pharmaceuticalcomposition as the CAR-expressing T cells, or may be prepared in aseparate pharmaceutical composition. Alternatively, the cytokine can beadministered shortly after administration of the CAR-expressing T cells,e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days afteradministration of the CAR-expressing T cells. In embodiments where thecytokine is administered in a dosing regimen that occurs over more thanone day, the first day of the cytokine dosing regimen can be on the sameday as administration with the CAR-expressing T cells, or the first dayof the cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days after administration of the CAR-expressing Tcells. In one embodiment, on the first day, the CAR-expressing T cellsare administered to the subject, and on the second day, a cytokine isadministered once a day for the next 7 days. In a preferred embodiment,the cytokine to be administered in combination with CAR-expressing Tcells is IL-7, IL-15, or IL-21.

In other embodiments, the cytokine is administered a period of timeafter administration of CAR-expressing cells, e.g., at least 2 weeks, 3weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or1 year or more after administration of CAR-expressing cells. In oneembodiment, the cytokine is administered after assessment of thesubject's response to the CAR-expressing cells. For example, the subjectis administered CAR-expressing cells according to the dosage andregimens described herein. The response of the subject to CAR-expressingcell therapy is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks,10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, or 1 year or more after administration ofCAR-expressing cells, using any of the methods described herein,including inhibition of tumor growth, reduction of circulating tumorcells, or tumor regression. Subjects that do not exhibit a sufficientresponse to CAR-expressing cell therapy can be administered a cytokine.Administration of the cytokine to the subject that has sub-optimalresponse to the CAR-expressing cell therapy improves CAR-expressing cellefficacy or anti-cancer activity. In a preferred embodiment, thecytokine administered after administration of CAR-expressing cells isIL-7.

Combination with CD19 Inhibitors

The methods and compositions disclosed herein can be used in combinationwith a CD19 inhibitor. In some embodiments, the CD123CAR-containingcells and the CD19 inhibitor (e.g., one or more cells that express a CARmolecule that binds CD19, e.g., a CAR molecule that binds CD19 describedherein) are administered simultaneously or concurrently, orsequentially.

In some embodiments, the CD123CAR-containing cells and the CD19inhibitor are infused into a subject simultaneously or concurrently,e.g., are admixed in the same infusion volume. For example, a populationof CD123CAR-containing cells and CD19CAR-containing cells are mixedtogether. Alternatively, a population of cells co-expressing a CD123CARand a CD19CAR is administered. In other embodiments, the simultaneousadministration comprises separate administration of theCD123CAR-containing cells and the CD19 inhibitor, e.g., within apredetermined time interval (e.g., within 15, 30, or 45 minutes of eachother).

In some embodiments, the start of the CD123CAR-containing cells and thestart of the CD19 inhibitor are within 1, 2, 3, 4, 6, 12, 18, or 24hours of each other, or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35,40, 60, 80, or 100 days of each other. In some embodiments, the end ofthe CD123CAR-containing cells delivery and the end of the CD19 inhibitordelivery are within 1, 2, 3, 4, 6, 12, 18, or 24 hours of each other, orwithin 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 60, 80, or 100 days ofeach other. In some embodiments, the overlap in terms of administrationbetween the of the CD123CAR-containing cells delivery (e.g., infusion)and the end of CD19 inhibitor delivery (e.g., infusion) is at least 1,2, 3, 4, 5, 10, 15, 20, 25, 30 minutes. In one embodiment, the CD19inhibitor is administered prior to the CD123CAR-containing cells. Inother embodiments, the CD123CAR-containing cells are administered priorto the CD19 inhibitor.

In some embodiments, the CD123CAR-containing cells are administeredwhile the CD19 inhibitor (e.g., one or more cells that express a CD19CARmolecule) is present (e.g., cells undergoing expansion) in the subject.In other embodiments, the CD19 inhibitor (e.g., one or more cells thatexpress a CD19CAR molecule) is administered while theCD123CAR-containing cells are present (e.g., cells undergoing expansion)in the subject.

A CD19 inhibitor includes, but is not limited to, a CD19 CAR-expressingcell, e.g., a CD19 CART cell, or an anti-CD19 antibody (e.g., ananti-CD19 mono- or bispecific antibody) or a fragment or conjugatethereof.

In one embodiment, a CAR-expressing cell described herein isadministered to a subject in combination with a CD19 CAR-cell (e.g.,CART cell) (e.g., CTL019, e.g., as described in WO2012/079000,incorporated herein by reference).

In other embodiments, the CAR-expressing cell described herein isadministered to a subject in combination with a CD19 CAR-cell (e.g.,CART cell) that includes a humanized antigen binding domain as describedin WO2014/153270 (e.g., Table 3 of WO2014/153270), incorporated hereinby reference.

The CD19 inhibitor (e.g., a first CD19 CAR-expressing cell) and a secondCD123 CAR-expressing cell may be expressed by the same cell type ordifferent types. For instance, in some embodiments, the cell expressinga CD19 CAR is a CD4+ T cell and the cell expressing a CD123 CAR is aCD8+ T cell, or the cell expressing a CD19 CAR is a CD8+ T cell and thecell expressing a CD123 CAR is a CD4+ T cell. In other embodiments, thecell expressing a CD19 CAR is a T cell and the cell expressing a CD123CAR is a NK cell, or the cell expressing a CD19 CAR is a NK cell and thecell expressing a CD123 CAR is a T cell. In other embodiments, the cellexpressing a CD19 CAR and the cell expressing a CD123 CAR are both NKcells or are both T cells, e.g., are both CD4+ T cells, or are both CD8+T cells. In yet other embodiments, a single cell expresses the CD19 CARand CD123 CAR, and this cell is, e.g., a NK cell or a T cell such as aCD4+ T cell or CD8+ T cell.

The first CAR and second CAR can comprise the same or differentintracellular signaling domains. For instance, in some embodiments, theCD19 CAR comprises a CD3 zeta signaling domain and the CD123 CARcomprises a costimulatory domain, e.g., a 41BB, CD27 or CD28costimulatory domain, while in some embodiments, the CD19 CAR comprisesa costimulatory domain, e.g., a 41BB, CD27 or CD28 costimulatory domainand the CD123 CAR comprises a CD3 zeta signaling domain. In otherembodiments, each of the CD19 CAR and the CD123 CAR comprises the sametype of primary signaling domain, e.g., a CD3 zeta signaling domain, butthe CD19 CAR and the CD123 CAR comprise different costimulatory domains,e.g., (1) the CD19 CAR comprises a 41BB costimulatory domain and theCD123 CAR comprises a different costimulatory domain e.g., a CD27costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatorydomain and the CD123 CAR comprises a different costimulatory domaine.g., a 41BB costimulatory domain, (3) the CD19 CAR comprises a 41BBcostimulatory domain and the CD123 CAR comprises a CD28 costimulatorydomain, (4) the CD19 CAR comprises a CD28 costimulatory domain and theCD123 CAR comprises a different costimulatory domain e.g., a 41BBcostimulatory domain, (5) the CD19 CAR comprises a CD27 costimulatorydomain and the CD123 CAR comprises a CD28 costimulatory domain, or (6)the CD19 CAR comprises a CD28 costimulatory domain and the CD123 CARcomprises a CD27 costimulatory domain. In another embodiment, a cellcomprises a CAR that comprises both a CD19 antigen-binding domain and aCD123 antigen-binding domain, e.g., a bispecific antibody.

In embodiments, the subject has acute myeloid leukemia (AML), e.g., aCD19 positive AML or a CD19 negative AML. In embodiments, the subjecthas a CD19+ lymphoma, e.g., a CD19+ Non-Hodgkin's Lymphoma (NHL), aCD19+ FL, or a CD19+ DLBCL. In embodiments, the subject has a relapsedor refractory CD19+ lymphoma. In embodiments, a lymphodepletingchemotherapy is administered to the subject prior to, concurrently with,or after administration (e.g., infusion) of CD19 CART cells. In anexample, the lymphodepleting chemotherapy is administered to the subjectprior to administration of CD19 CART cells. For example, thelymphodepleting chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4 days)prior to CD19 CART cell infusion. In embodiments, multiple doses of CD19CART cells are administered, e.g., as described herein. For example, asingle dose comprises about 5×10⁸ CD19 CART cells. In embodiments, alymphodepleting chemotherapy is administered to the subject prior to,concurrently with, or after administration (e.g., infusion) of aCAR-expressing cell described herein, e.g., a non-CD19 CAR-expresingcell. In embodiments, a CD19 CART is administered to the subject priorto, concurrently with, or after administration (e.g., infusion) of anon-CD19 CAR-expressing cell, e.g., a non-CD19 CAR-expressing celldescribed herein.

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a CD19 CAR-expressingcell, e.g., CTL019, e.g., as described in WO2012/079000, incorporatedherein by reference, for treatment of a disease associated with theexpression of CD123, e.g., a cancer described herein. Without beingbound by theory, it is believed that administering a CD19 CAR-expressingcell in combination with a CAR-expressing cell improves the efficacy ofa CAR-expressing cell described herein by targeting early lineage cancercells, e.g., cancer stem cells, modulating the immune response,depleting regulatory B cells, and/or improving the tumormicroenvironment. For example, a CD19 CAR-expressing cell targets cancercells that express early lineage markers, e.g., cancer stem cells andCD19-expressing cells, while the CAR-expressing cell described hereintargets cancer cells that express later lineage markers, e.g., CD123.This preconditioning approach can improve the efficacy of theCAR-expressing cell described herein. In such embodiments, the CD19CAR-expressing cell is administered prior to, concurrently with, orafter administration (e.g., infusion) of a CAR-expressing cell describedherein.

In embodiments, a CAR-expressing cell described herein also expresses aCAR targeting CD19, e.g., a CD19 CAR. In an embodiment, the cellexpressing a CAR described herein and a CD19 CAR is administered to asubject for treatment of a cancer described herein, e.g., AML. In anembodiment, the configurations of one or both of the CAR moleculescomprise a primary intracellular signaling domain and a costimulatorysignaling domain. In another embodiment, the configurations of one orboth of the CAR molecules comprise a primary intracellular signalingdomain and two or more, e.g., 2, 3, 4, or 5 or more, costimulatorysignaling domains. In such embodiments, the CAR molecule describedherein and the CD19 CAR may have the same or a different primaryintracellular signaling domain, the same or different costimulatorysignaling domains, or the same number or a different number ofcostimulatory signaling domains. Alternatively, the CAR described hereinand the CD19 CAR are configured as a split CAR, in which one of the CARmolecules comprises an antigen binding domain and a costimulatory domain(e.g., 4-1BB), while the other CAR molecule comprises an antigen bindingdomain and a primary intracellular signaling domain (e.g., CD3 zeta).

In an embodiment, the CAR described herein and the second CAR, e.g.,CD19 CAR, are on the same vector or are on two different vectors. Inembodiments where the CAR described herein and the second CAR, e.g.,CD19 CAR, are on the same vector, the nucleic acid sequences encodingthe CAR described herein and the second CAR, e.g., CD19 CAR are in thesame frame, and are separated by one or more peptide cleavage sites,e.g., P2A.

In other embodiments, the CAR-expressing cell disclosed herein isadministered in combination with an anti-CD19 antibody inhibitor. In oneembodiment, the anti-CD19 antibody is a humanized antigen binding domainas described in WO2014/153270 (e.g., Table 3 of WO2014/153270)incorporated herein by reference, or a conjugate thereof. Otherexemplary anti-CD19 antibodies or fragments or conjugates thereof,include but are not limited to, blinatumomab, SAR3419 (Sanofi), MEDI-551(MedImmune LLC), Combotox, DT2219ARL (Masonic Cancer Center), MOR-208(also called XmAb-5574; MorphoSys), XmAb-5871 (Xencor), MDX-1342(Bristol-Myers Squibb), SGN-CD19A (Seattle Genetics), and AFM11 (AffimedTherapeutics). See, e.g., Hammer. MAbs. 4.5(2012): 571-77. Blinatomomabis a bispecific antibody comprised of two scFvs—one that binds to CD19and one that binds to CD3. Blinatomomab directs T cells to attack cancercells. See, e.g., Hammer et al.; Clinical Trial Identifier No.NCT00274742 and NCT01209286. MEDI-551 is a humanized anti-CD19 antibodywith a Fc engineered to have enhanced antibody-dependent cell-mediatedcytotoxicity (ADCC). See, e.g., Hammer et al.; and Clinical TrialIdentifier No. NCT01957579. Combotox is a mixture of immunotoxins thatbind to CD19 and CD22. The immunotoxins are made up of scFv antibodyfragments fused to a deglycosylated ricin A chain. See, e.g., Hammer etal.; and Herrera et al. J. Pediatr. Hematol. Oncol. 31.12(2009):936-41;Schindler et al. Br. J. Haematol. 154.4(2011):471-6. DT2219ARL is abispecific immunotoxin targeting CD19 and CD22, comprising two scFvs anda truncated diphtheria toxin. See, e.g., Hammer et al.; and ClinicalTrial Identifier No. NCT00889408. SGN-CD19A is an antibody-drugconjugate (ADC) comprised of an anti-CD19 humanized monoclonal antibodylinked to a synthetic cytotoxic cell-killing agent, monomethylauristatin F (MMAF). See, e.g., Hammer et al.; and Clinical TrialIdentifier Nos. NCT01786096 and NCT01786135. SAR3419 is an anti-CD19antibody-drug conjugate (ADC) comprising an anti-CD19 humanizedmonoclonal antibody conjugated to a maytansine derivative via acleavable linker. See, e.g., Younes et al, J. Clin. Oncol, 30.2(2012):2776-82; Hammer et al.; Clinical Trial Identifier No. NCT00549185: andBlanc et al. Clin Cancer Res. 2011; 17:6448-58. XmAb-5871 is anFc-engineered, humanized anti-CD19 antibody. See, e.g., Hammer et al.MDX-1342 is a human Fc-engineered anti-CD19 antibody with enhanced ADCC.See, e.g., Hammer et al. In embodiments, the antibody molecule is abispecific anti-CD19 and anti-CD3 molecule. For instance, AFM11 is abispecific antibody that targets CD19 and CD3. See, e.g., Hammer et al.;and Clinical Trial Identifier No. NCT02106091. In some embodiments, ananti-CD19 antibody described herein is conjugated or otherwise bound toa therapeutic agent, e.g., a chemotherapeutic agent, peptide vaccine(such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971),immunosuppressive agent, or immunoablative agent, e.g., cyclosporin,azathioprine, methotrexate, mycophenolate, FK506, CAMPATH, anti-CD3antibody, cytoxin, fludarabine, rapamycin, mycophenolic acid, steroid,FR901228, or cytokine.

Combination with a Low Dose of an mTOR Inhibitor

Methods described herein use low, immune enhancing, doses of mTORinhibitors, e.g., allosteric mTOR inhibitors, including rapalogs such asRAD001. Administration of a low, immune enhancing, dose of an mTORinhibitor (e.g., a dose that is insufficient to completely suppress theimmune system, but sufficient to improve immune function) can optimizethe performance of immune effector cells, e.g., T cells orCAR-expressing cells, in the subject. Methods for measuring mTORinhibition, dosages, treatment regimens, and suitable pharmaceuticalcompositions are described in U.S. Patent Application No. 2015/01240036,hereby incorporated by reference.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor can result in one or more of the following:

-   -   i) a decrease in the number of PD-1 positive immune effector        cells;    -   ii) an increase in the number of PD-1 negative immune effector        cells;    -   iii) an increase in the ratio of PD-1 negative immune effector        cells/PD-1 positive immune effector cells;    -   iv) an increase in the number of naive T cells;    -   v) an increase in the expression of one or more of the following        markers: CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on        memory T cells, e.g., memory T cell precursors;    -   vi) a decrease in the expression of KLRG1, e.g., on memory T        cells, e.g., memory T cell precursors; or    -   vii) an increase in the number of memory T cell precursors,        e.g., cells with any one or combination of the following        characteristics: increased CD62L^(high) increased CD127^(high),        increased CD27⁺, decreased KLRG1, and increased BCL2;        and wherein any of the foregoing, e.g., i), ii), iii), iv), v),        vi), or vii), occurs e.g., at least transiently, e.g., as        compared to a non-treated subject.

In another embodiment, administration of a low, immune enhancing, doseof an mTOR inhibitor results in increased or prolonged proliferation ofCAR-expressing cells, e.g., in culture or in a subject, e.g., ascompared to non-treated CAR-expressing cells or a non-treated subject.In embodiments, increased proliferation is associated with in anincrease in the number of CAR-expressing cells. Methods for measuringincreased or prolonged proliferation are described in Examples 9 and 10.In another embodiment, administration of a low, immune enhancing, doseof an mTOR inhibitor results in increased killing of cancer cells byCAR-expressing cells, e.g., in culture or in a subject, e.g., ascompared to non-treated CAR-expressing cells or a non-treated subject.In embodiments, increased killing of cancer cells is associated with ina decrease in tumor volume. Methods for measuring increased killing ofcancer cells are described in Example 2.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor, e.g., an allosteric mTORinhibitor, e.g., RAD001, or a catalytic mTOR inhibitor. For example,administration of the low, immune enhancing, dose of the mTOR inhibitorcan be initiated prior to administration of a CAR-expressing celldescribed herein; completed prior to administration of a CAR-expressingcell described herein; initiated at the same time as administration of aCAR-expressing cell described herein; overlapping with administration ofa CAR-expressing cell described herein; or continuing afteradministration of a CAR-expressing cell described herein.

Alternatively or in addition, administration of a low, immune enhancing,dose of an mTOR inhibitor can optimize immune effector cells to beengineered to express a CAR molecule described herein. In suchembodiments, administration of a low, immune enhancing, dose of an mTORinhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalyticinhibitor, is initiated or completed prior to harvest of immune effectorcells, e.g., T cells or NK cells, to be engineered to express a CARmolecule described herein, from a subject.

In another embodiment, immune effector cells, e.g., T cells or NK cells,to be engineered to express a CAR molecule described herein, e.g., afterharvest from a subject, or CAR-expressing immune effector cells, e.g., Tcells or NK cells, e.g., prior to administration to a subject, can becultured in the presence of a low, immune enhancing, dose of an mTORinhibitor.

In an embodiment, administering to the subject a low, immune enhancing,dose of an mTOR inhibitor comprises administering, e.g., once per week,e.g., in an immediate release dosage form, 0.1 to 20, 0.5 to 10, 2.5 to7.5, 3 to 6, or about 5, mgs of RAD001, or a bioequivalent dose thereof.In an embodiment, administering to the subject a low, immune enhancing,dose of an mTOR inhibitor comprises administering, e.g., once per week,e.g., in a sustained release dosage form, 0.3 to 60, 1.5 to 30, 7.5 to22.5, 9 to 18, or about 15 mgs of RAD001, or a bioequivalent dosethereof.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 90%, at least10 but no more than 90%, at least 15, but no more than 90%, at least 20but no more than 90%, at least 30 but no more than 90%, at least 40 butno more than 90%, at least 50 but no more than 90%, at least 60 but nomore than 90%, at least 70 but no more than 90%, at least 5 but no morethan 80%, at least 10 but no more than 80%, at least 15, but no morethan 80%, at least 20 but no more than 80%, at least 30 but no more than80%, at least 40 but no more than 80%, at least 50 but no more than 80%,at least 60 but no more than 80%, at least 5 but no more than 70%, atleast 10 but no more than 70%, at least 15, but no more than 70%, atleast 20 but no more than 70%, at least 30 but no more than 70%, atleast 40 but no more than 70%, at least 50 but no more than 70%, atleast 5 but no more than 60%, at least 10 but no more than 60%, at least15, but no more than 60%, at least 20 but no more than 60%, at least 30but no more than 60%, at least 40 but no more than 60%, at least 5 butno more than 50%, at least 10 but no more than 50%, at least 15, but nomore than 50%, at least 20 but no more than 50%, at least 30 but no morethan 50%, at least 40 but no more than 50%, at least 5 but no more than40%, at least 10 but no more than 40%, at least 15, but no more than40%, at least 20 but no more than 40%, at least 30 but no more than 40%,at least 35 but no more than 40%, at least 5 but no more than 30%, atleast 10 but no more than 30%, at least 15, but no more than 30%, atleast 20 but no more than 30%, or at least 25 but no more than 30%.

The extent of mTOR inhibition can be conveyed as, or corresponds to, theextent of P70 S6 kinase inhibition, e.g., the extent of mTOR inhibitioncan be determined by the level of decrease in P70 S6 kinase activity,e.g., by the decrease in phosphorylation of a P70 S6 kinase substrate.The level of mTOR inhibition can be evaluated by various methods, suchas measuring P70 S6 kinase activity by the Boulay assay, as described inU.S. Patent Application No. 2015/01240036, hereby incorporated byreference, or as described in U.S. Pat. No. 7,727,950, herebyincorporated by reference; measuring the level of phosphorylated S6 bywestern blot; or evaluating a change in the ratio of PD1 negative immuneeffector cells to PD1 positive immune effector cells.

As used herein, the term “mTOR inhibitor” refers to a compound orligand, or a pharmaceutically acceptable salt thereof, which inhibitsthe mTOR kinase in a cell. In an embodiment, an mTOR inhibitor is anallosteric inhibitor. Allosteric mTOR inhibitors include the neutraltricyclic compound rapamycin (sirolimus), rapamycin-related compounds,that is compounds having structural and functional similarity torapamycin including, e.g., rapamycin derivatives, rapamycin analogs(also referred to as rapalogs) and other macrolide compounds thatinhibit mTOR activity. In an embodiment, an mTOR inhibitor is acatalytic inhibitor.

Rapamycin is a known macrolide antibiotic produced by Streptomyceshygroscopicus having the structure shown in Formula A.

See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688;Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat.No. 3,929,992. There are various numbering schemes proposed forrapamycin. To avoid confusion, when specific rapamycin analogs are namedherein, the names are given with reference to rapamycin using thenumbering scheme of formula A.

Rapamycin analogs useful in the invention are, for example,0-substituted analogs in which the hydroxyl group on the cyclohexyl ringof rapamycin is replaced by OR₁ in which R₁ is hydroxyalkyl,hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; e.g. RAD001, alsoknown as, everolimus as described in U.S. Pat. No. 5,665,772 andWO94/09010 the contents of which are incorporated by reference. Othersuitable rapamycin analogs include those substituted at the 26- or28-position. The rapamycin analog may be an epimer of an analogmentioned above, particularly an epimer of an analog substituted inposition 40, 28 or 26, and may optionally be further hydrogenated, e.g.as described in U.S. Pat. No. 6,015,815, WO95/14023 and WO99/15530 thecontents of which are incorporated by reference, e.g. ABT578 also knownas zotarolimus or a rapamycin analog described in U.S. Pat. No.7,091,213, WO98/02441 and WO01/14387 the contents of which areincorporated by reference, e.g. AP23573 also known as ridaforolimus.

Examples of rapamycin analogs suitable for use in the present inventionfrom U.S. Pat. No. 5,665,772 include, but are not limited to,40-O-benzyl-rapamycin, 40-O-(4′-hydroxymethyl)benzyl-rapamycin,40-O-[4′-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin,40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′E,4′S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,40-O-(6-hydroxy)hexyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin,40-O-[(35)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(25)-2,3-dihydroxyprop-1-yl]-rapamycin,40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin,40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,40-O-[2-(N-methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin,39-O-desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,40-O-(2-nicotinamidoethyl)-rapamycin,40-O-(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-tolylsulfonamidoethylkapamycin and40-O-[2-(4′,5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin.

Other rapamycin analogs useful in the present invention are analogswhere the hydroxyl group on the cyclohexyl ring of rapamycin and/or thehydroxy group at the 28 position is replaced with an hydroxyester groupare known, for example, rapamycin analogs found in U.S. Pat. No.RE44,768, e.g. temsirolimus.

Other rapamycin analogs useful in the preset invention include thosewherein the methoxy group at the 16 position is replaced with anothersubstituent, preferably (optionally hydroxy-substituted) alkynyloxy,benzyl, orthomethoxybenzyl or chlorobenzyl and/or wherein the mexthoxygroup at the 39 position is deleted together with the 39 carbon so thatthe cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the39 position methyoxy group; e.g. as described in WO95/16691 andWO96/41807 the contents of which are incorporated by reference. Theanalogs can be further modified such that the hydroxy at the 40-positionof rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to,16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,16-demthoxy-16-(propargyl)oxy-rapamycin,16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,16-demthoxy-16-benzyloxy-rapamycin,16-demethoxy-16-ortho-methoxybenzyl-rapamycin,16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to,32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin,16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable rapamycin analog is umirolimus as described inUS2005/0101624 the contents of which are incorporated by reference.

RAD001, otherwise known as everolimus (Afinitor®), has the chemical name(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone

Further examples of allosteric mTOR inhibitors include sirolimus(rapamycin, AY-22989),40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (alsocalled temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669).Other examples of allosteric mTor inhibtors include zotarolimus (ABT578)and umirolimus.

Alternatively or additionally, catalytic, ATP-competitive mTORinhibitors have been found to target the mTOR kinase domain directly andtarget both mTORC1 and mTORC2. These are also more effective inhibitorsof mTORC1 than such allosteric mTOR inhibitors as rapamycin, becausethey modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile,or the monotosylate salt form. the synthesis of BEZ235 is described inWO2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C. M., etal., Cancer Res, 2010, 70(1), 288-298) which has the chemical name{5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-methoxyphenyl}-methanol;3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide(WO09104019);3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine(WO10051043 and WO2013023184); AN-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide(WO07044729 and WO12006552); PKI-587 (Venkatesan, A. M., J. Med. Chem.,2010, 53, 2636-2645) which has the chemical name1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea;GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43) which has thechemical name2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide;5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(WO10114484);(E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamide(WO12007926).

Further examples of catalytic mTOR inhibitors include8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one(WO2006/122806) and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J.,2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammaliantarget of rapamycin (mTOR).) WYE-354 is another example of a catalyticmTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivoActivity of Novel ATP-Competitive and Selective Inhibitors of theMammalian Target of Rapamycin. Cancer Res. 69(15): 6232-6240).

mTOR inhibitors useful according to the present invention also includeprodrugs, derivatives, pharmaceutically acceptable salts, or analogsthereof of any of the foregoing.

mTOR inhibitors, such as RAD001, may be formulated for delivery based onwell-established methods in the art based on the particular dosagesdescribed herein. In particular, U.S. Pat. No. 6,004,973 (incorporatedherein by reference) provides examples of formulations useable with themTOR inhibitors described herein.

Methods and Biomarkers for Evaluating CAR-Effectiveness or SampleSuitability

In another aspect, the invention features a method of evaluating ormonitoring the effectiveness of a CAR-expressing cell therapy (e.g., aCD123 CAR therapy), in a subject (e.g., a subject having a cancer, e.g.,a hematological cancer), or the suitability of a sample (e.g., anapheresis sample) for a CAR therapy (e.g., a CD123 CAR therapy). Themethod includes acquiring a value of effectiveness to the CAR therapy,or sample suitability, wherein said value is indicative of theeffectiveness or suitability of the CAR-expressing cell therapy.

In embodiments, the value of effectiveness to the CAR therapy, or samplesuitability, comprises a measure of one, two, three, four, five, six ormore (all) of the following:

(i) the level or activity of one, two, three, or more (e.g., all) ofresting T_(EFF) cells, resting T_(REG) cells, younger T cells (e.g.,younger CD4 or CD8 cells, or gamma/delta T cells), or early memory Tcells, or a combination thereof, in a sample (e.g., an apheresis sampleor a manufactured CAR-expressing cell product sample);

(ii) the level or activity of one, two, three, or more (e.g., all) ofactivated T_(EFF) cells, activated T_(REG) cells, older T cells (e.g.,older CD4 or CD8 cells), or late memory T cells, or a combinationthereof, in a sample (e.g., an apheresis sample or a manufacturedCAR-expressing cell product sample);

(iii) the level or activity of an immune cell exhaustion marker, e.g.,one, two or more immune checkpoint inhibitors (e.g., PD-1, PD-L1, TIM-3and/or LAG-3) in a sample (e.g., an apheresis sample or a manufacturedCAR-expressing cell product sample). In one embodiment, an immune cellhas an exhausted phenotype, e.g., co-expresses at least two exhaustionmarkers, e.g., co-expresses PD-1 and TIM-3. In other embodiments, animmune cell has an exhausted phenotype, e.g., co-expresses at least twoexhaustion markers, e.g., co-expresses PD-1 and LAG-3;

(iv) the level or activity of CD27 and/or CD45RO− (e.g., CD27+ CD45RO−)immune effector cells, e.g., in a CD4+ or a CD8+ T cell population, in asample (e.g., an apheresis sample or a manufactured CAR-expressing cellproduct sample);

(v) the level or activity of one, two, three, four, five, ten, twenty ormore of the biomarkers chosen from CCL20, IL-17a and/or IL-6, PD-1,PD-L1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1;

(vi) a cytokine level or activity (e.g., quality of cytokine repertoire)in a CAR-expressing cell product sample, e.g., CD123-expressing cellproduct sample; or

(vii) a transduction efficiency of a CAR-expressing cell in amanufactured CAR-expressing cell product sample.

In some embodiments of any of the methods disclosed herein, theCAR-expressing cell therapy comprises a plurality (e.g., a population)of CAR-expressing immune effector cells, e.g., a plurality (e.g., apopulation) of T cells or NK cells, or a combination thereof. In oneembodiment, the CAR-expressing cell therapy is a CD123 CAR therapy.

In some embodiments of any of the methods disclosed herein, the measureof one or more of (i)-(vii) is obtained from an apheresis sampleacquired from the subject. The apheresis sample can be evaluated priorto infusion or re-infusion.

In some embodiments of any of the methods disclosed herein, the measureof one or more of (i)-(vii) is obtained from a manufacturedCAR-expressing cell product sample, e.g., CD123 CAR-expressing cellproduct sample. The manufactured CAR-expressing cell product can beevaluated prior to infusion or re-infusion.

In some embodiments of any of the methods disclosed herein, the subjectis evaluated prior to receiving, during, or after receiving, theCAR-expressing cell therapy.

In some embodiments of any of the methods disclosed herein, the measureof one or more of (i)-(vii) evaluates a profile for one or more of geneexpression, flow cytometry or protein expression.

In some embodiments of any of the methods disclosed herein, the methodfurther comprises identifying the subject as a responder, anon-responder, a relapser or a non-relapser, based on a measure of oneor more of (i)-(vii).

In some embodiments of any of the methods disclosed herein, a responder(e.g., a complete responder) has, or is identified as having, a greaterlevel or activity of one, two, or more (all) of GZMK, PPF1BP2, or naïveT cells as compared to a non-responder.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater level oractivity of one, two, three, four, five, six, seven, or more (e.g., all)of IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22, effector T cells, orregulatory T cells, as compared to a responder.

In an embodiment, a relapser is a patient having, or who is identifiedas having, an increased level of expression of one or more of (e.g., 2,3, 4, or all of) the following genes, compared to non relapsers:MIR199A1, MIR1203, uc021ovp, ITM2C, and HLA-DQB1 and/or a decreasedlevels of expression of one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or all of) the following genes, compared to non relapsers:PPIAL4D, TTTY10, TXLNG2P, MIR4650-1, KDM5D, USP9Y, PRKY, RPS4Y2, RPS4Y1,NCRNA00185, SULT1E1, and EIF1AY.

In some embodiments of any of the methods disclosed herein, a completeresponder has, or is identified as having, a greater, e.g., astatistically significant greater, percentage of CD8+ T cells comparedto a reference value, e.g., a non-responder percentage of CD8+ T cells.

In some embodiments of any of the methods disclosed herein, a completeresponder has, or is identified as having, a greater percentage of CD27+CD45RO− immune effector cells, e.g., in the CD8+ population, compared toa reference value, e.g., a non-responder number of CD27+ CD45RO− immuneeffector cells.

In some embodiments of any of the methods disclosed herein, a completeresponder or a partial responder has, or is identified as having, agreater, e.g., a statistically significant greater, percentage of CD4+ Tcells compared to a reference value, e.g., a non-responder percentage ofCD4+ T cells.

In some embodiments of any of the methods disclosed herein, a completeresponder has, or is identified as having, a greater percentage of one,two, three, or more (e.g., all) of resting T_(EFF) cells, restingT_(REG) cells, younger T cells (e.g., younger CD4 or CD8 cells, orgamma/delta T cells), or early memory T cells, or a combination thereof,compared to a reference value, e.g., a non-responder number of restingT_(EFF) cells, resting T_(REG) cells, younger T cells (e.g., younger CD4or CD8 cells), or early memory T cells.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofone, two, three, or more (e.g., all) of activated T_(EFF) cells,activated T_(REG) cells, older T cells (e.g., older CD4 or CD8 cells),or late memory T cells, or a combination thereof, compared to areference value, e.g., a responder number of activated T_(EFF) cells,activated T_(REG) cells, older T cells (e.g., older CD4 or CD8 cells),or late memory T cells.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofan immune cell exhaustion marker, e.g., one, two or more immunecheckpoint inhibitors (e.g., PD-1, PD-L1, TIM-3 and/or LAG-3). In oneembodiment, a non-responder has, or is identified as having, a greaterpercentage of PD-1, PD-L1, or LAG-3 expressing immune effector cells(e.g., CD4+ T cells and/or CD8+ T cells) (e.g., CAR-expressing CD4+cells and/or CD8+ T cells) compared to the percentage of PD-1 or LAG-3expressing immune effector cells from a responder.

In one embodiment, a non-responder has, or is identified as having, agreater percentage of immune cells having an exhausted phenotype, e.g.,immune cells that co-express at least two exhaustion markers, e.g.,co-expresses PD-1, PD-L1 and/or TIM-3. In other embodiments, anon-responder has, or is identified as having, a greater percentage ofimmune cells having an exhausted phenotype, e.g., immune cells thatco-express at least two exhaustion markers, e.g., co-expresses PD-1 andLAG-3.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofPD-1/PD-L1+/LAG-3+ cells in the CAR-expressing cell population (e.g., aCD123 CAR+ cell population) compared to a responder (e.g., a completeresponder) to the CAR-expressing cell therapy.

In some embodiments of any of the methods disclosed herein, a partialresponder has, or is identified as having, a higher percentages ofPD-1/PD-L1+/LAG-3+ cells, than a responder, in the CAR-expressing cellpopulation (e.g., a CD123 CAR+ cell population).

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, an exhausted phenotype ofPD1/PD-L1+CAR+ and co-expression of LAG3 in the CAR-expressing cellpopulation (e.g., a CD123 CAR+ cell population).

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofPD-1/PD-L1+/TIM-3+ cells in the CAR-expressing cell population (e.g., aCD123 CAR+ cell population) compared to the responder (e.g., a completeresponder).

In some embodiments of any of the methods disclosed herein, a partialresponders has, or is identified as having, a higher percentage ofPD-1/PD-L1+/TIM-3+ cells, than responders, in the CAR-expressing cellpopulation (e.g., a CD123 CAR+ cell population).

In some embodiments of any of the methods disclosed herein, the presenceof CD8+CD27+ CD45RO− T cells in an apheresis sample is a positivepredictor of the subject response to a CAR-expressing cell therapy(e.g., a CD123 CAR therapy).

In some embodiments of any of the methods disclosed herein, a highpercentage of PD1+CAR+ and LAG3+ or TIM3+ T cells in an apheresis sampleis a poor prognostic predictor of the subject response to aCAR-expressing cell therapy (e.g., a CD123 CAR therapy).

In some embodiments of any of the methods disclosed herein, theresponder (e.g., the complete or partial responder) has one, two, threeor more (or all) of the following profile:

(i) has a greater number of CD27+ immune effector cells compared to areference value, e.g., a non-responder number of CD27+ immune effectorcells;

(ii) (i) has a greater number of CD8+ T cells compared to a referencevalue, e.g., a non-responder number of CD8+ T cells;

(iii) has a lower number of immune cells expressing one or morecheckpoint inhibitors, e.g., a checkpoint inhibitor chosen from PD-1,PD-L1, LAG-3, TIM-3, or KLRG-1, or a combination, compared to areference value, e.g., a non-responder number of cells expressing one ormore checkpoint inhibitors; or

(iv) has a greater number of one, two, three, four or more (all) ofresting T_(EFF) cells, resting T_(REG) cells, naïve CD4 cells,unstimulated memory cells or early memory T cells, or a combinationthereof, compared to a reference value, e.g., a non-responder number ofresting T_(EFF) cells, resting T_(REG) cells, naïve CD4 cells,unstimulated memory cells or early memory T cells.

In some embodiments of any of the methods disclosed herein, the cytokinelevel or activity of (vi) is chosen from one, two, three, four, five,six, seven, eight, or more (or all) of cytokine CCL20/MIP3a, IL17A, IL6,GM-CSF, IFNγ, IL10, IL13, IL2, IL21, IL4, IL5, IL9 or TNFα, or acombination thereof. The cytokine can be chosen from one, two, three,four or more (all) of IL-17a, CCL20, IL2, IL6, or TNFa. In oneembodiment, an increased level or activity of a cytokine is chosen fromone or both of IL-17a and CCL20, is indicative of increasedresponsiveness or decreased relapse.

In some embodiments of any of the methods disclosed herein, atransduction efficiency of 15% or higher in (vii) is indicative ofincreased responsiveness or decreased relapse.

In some embodiments of any of the methods disclosed herein, atransduction efficiency of less than 15% in (vii) is indicative ofdecreased responsiveness or increased relapse.

In embodiments, the responder, a non-responder, a relapser or anon-relapser identified by the methods herein can be further evaluatedaccording to clinical criteria. For example, a complete responder has,or is identified as, a subject having a disease, e.g., a cancer, whoexhibits a complete response, e.g., a complete remission, to atreatment. A complete response may be identified, e.g., using the NCCNGuidelines®, or Cheson et al, J Clin Oncol 17:1244 (1999) and Cheson etal., “Revised Response Criteria for Malignant Lymphoma”, J Clin Oncol25:579-586 (2007) (both of which are incorporated by reference herein intheir entireties), as described herein. A partial responder has, or isidentified as, a subject having a disease, e.g., a cancer, who exhibitsa partial response, e.g., a partial remission, to a treatment. A partialresponse may be identified, e.g., using the NCCN Guidelines®, or Chesoncriteria as described herein. A non-responder has, or is identified as,a subject having a disease, e.g., a cancer, who does not exhibit aresponse to a treatment, e.g., the patient has stable disease orprogressive disease. A non-responder may be identified, e.g., using theNCCN Guidelines®, or Cheson criteria as described herein.

Alternatively, or in combination with the methods disclosed herein,responsive to said value, performing one, two, three, four or more of:

administering e.g., to a responder or a non-relapser, a CAR-expressingcell therapy;

administered an altered dosing of a CAR-expressing cell therapy;

altering the schedule or time course of a CAR-expressing cell therapy;

administering, e.g., to a non-responder or a partial responder, anadditional agent in combination with a CAR-expressing cell therapy,e.g., a checkpoint inhibitor, e.g., a checkpoint inhibitor describedherein;

administering to a non-responder or partial responder a therapy thatincreases the number of younger T cells in the subject prior totreatment with a CAR-expressing cell therapy;

modifying a manufacturing process of a CAR-expressing cell therapy,e.g., enriching for younger T cells prior to introducing a nucleic acidencoding a CAR, or increasing the transduction efficiency, e.g., for asubject identified as a non-responder or a partial responder;

administering an alternative therapy, e.g., for a non-responder orpartial responder or relapser; or

if the subject is, or is identified as, a non-responder or a relapser,decreasing the T_(REG) cell population and/or T_(REG) gene signature,e.g., by one or more of CD25 depletion, administration ofcyclophosphamide, anti-GITR antibody, or a combination thereof.

In certain embodiments, the subject is pre-treated with an anti-GITRantibody. In certain embodiment, the subject is treated with ananti-GITR antibody prior to infusion or re-infusion.

Biopolymer Delivery Methods

In some embodiments, one or more CAR-expressing cells as disclosedherein can be administered or delivered to the subject via a biopolymerscaffold, e.g., a biopolymer implant. Biopolymer scaffolds can supportor enhance the delivery, expansion, and/or dispersion of theCAR-expressing cells described herein. A biopolymer scaffold comprises abiocompatible (e.g., does not substantially induce an inflammatory orimmune response) and/or a biodegradable polymer that can be naturallyoccurring or synthetic.

Examples of suitable biopolymers include, but are not limited to, agar,agarose, alginate, alginate/calcium phosphate cement (CPC),beta-galactosidase ((3-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose),cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acidcollagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate)(PHBHHx), poly(lactide), poly(caprolactone) (PCL),poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO),poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO),polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate,alone or in combination with any other polymer composition, in anyconcentration and in any ratio. The biopolymer can be augmented ormodified with adhesion- or migration-promoting molecules, e.g.,collagen-mimetic peptides that bind to the collagen receptor oflymphocytes, and/or stimulatory molecules to enhance the delivery,expansion, or function, e.g., anti-cancer activity, of the cells to bedelivered. The biopolymer scaffold can be an injectable, e.g., a gel ora semi-solid, or a solid composition.

In some embodiments, CAR-expressing cells described herein are seededonto the biopolymer scaffold prior to delivery to the subject. Inembodiments, the biopolymer scaffold further comprises one or moreadditional therapeutic agents described herein (e.g., anotherCAR-expressing cell, an antibody, or a small molecule) or agents thatenhance the activity of a CAR-expressing cell, e.g., incorporated orconjugated to the biopolymers of the scaffold. In embodiments, thebiopolymer scaffold is injected, e.g., intratumorally, or surgicallyimplanted at the tumor or within a proximity of the tumor sufficient tomediate an anti-tumor effect. Additional examples of biopolymercompositions and methods for their delivery are described in Stephan etal., Nature Biotechnology, 2015, 33:97-101; and WO2014/110591.

Pharmaceutical Compositions and Treatments

Pharmaceutical compositions of the present invention may comprise aCAR-expressing cell, e.g., a plurality of CAR-expressing cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶cells/kg body weight, including all integer values within those ranges.T cell compositions may also be administered multiple times at thesedosages.

In some embodiments, a dose of CAR cells (e.g., CD123 CAR cells)comprises about 1×10⁶, 1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷,2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, or 5×10⁸ cells/kg. In some embodiments, adose of CAR cells (e.g., CD123 CAR cells) comprises at least about1×10⁶, 1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷,1×10⁸, 2×10⁸, or 5×10⁸ cells/kg. In some embodiments, a dose of CARcells (e.g., CD123 CAR cells) comprises up to about 1×10⁶, 1.1×10⁶,2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, or5×10⁸ cells/kg. In some embodiments, a dose of CAR cells (e.g., CD123CAR cells) comprises about 1.1×10⁶-1.8×10⁷ cells/kg. In someembodiments, a dose of CAR cells (e.g., CD123 CAR cells) comprises about1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells.In some embodiments, a dose of CAR cells (e.g., CD123 CAR cells)comprises at least about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸,1×10⁹, 2×10⁹, or 5×10⁹ cells. In some embodiments, a dose of CAR cells(e.g., CD123 CAR cells) comprises up to about 1×10⁷, 2×10⁷, 5×10⁷,1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells.

The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988).

In certain aspects, it may be desired to administer activated T cells toa subject and then subsequently redraw blood (or have an apheresisperformed), activate T cells therefrom according to the presentinvention, and reinfuse the patient with these activated and expanded Tcells. This process can be carried out multiple times every few weeks.In certain aspects, T cells can be activated from blood draws of from 10cc to 400 cc. In certain aspects, T cells are activated from blood drawsof 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the CAR-expressing cell (e.g., T cellor NK cell) compositions of the present invention are administered to apatient by intradermal or subcutaneous injection. In one aspect, theCAR-expressing cell (e.g., T cell or NK cell) compositions of thepresent invention are administered by i.v. injection. The compositionsof the CAR-expressing cell (e.g., T cell or NK cell) may be injecteddirectly into a tumor, lymph node, or site of infection.

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., immune effector cells(e.g., T cells or NK cells). These immune effector cell (e.g., T cell orNK cell) isolates may be expanded by methods known in the art andtreated such that one or more CAR constructs of the invention may beintroduced, thereby creating a CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) of the invention. Subjects in need thereof maysubsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. In certainaspects, following or concurrent with the transplant, subjects receivean infusion of the expanded CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) of the present invention. In an additionalaspect, expanded cells are administered before or following surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

In one embodiment, the CAR is introduced into immune effector cells(e.g., T cells or NK cells), e.g., using in vitro transcription, and thesubject (e.g., human) receives an initial administration of aCAR-expressing cell (e.g., CAR T cell or CAR-expressing NK cell) of theinvention, and one or more subsequent administrations of theCAR-expressing cell (e.g., CAR T cell or CAR-expressing NK cell) of theinvention, wherein the one or more subsequent administrations areadministered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, or 2 days after the previous administration. In one embodiment,more than one administration of the CAR-expressing cell (e.g., CAR Tcell or CAR-expressing NK cell) of the invention are administered to thesubject (e.g., human) per week, e.g., 2, 3, or 4 administrations of theCAR-expressing cell (e.g., CAR T cell or CAR-expressing NK cell) of theinvention are administered per week. In one embodiment, the subject(e.g., human subject) receives more than one administration of theCAR-expressing cell (e.g., CAR T cell or CAR-expressing NK cell) perweek (e.g., 2, 3 or 4 administrations per week) (also referred to hereinas a cycle), followed by a week of no CAR-expressing cell (e.g., CAR Tcell or CAR-expressing NK cell) administrations, and then one or moreadditional administration of the CAR-expressing cell (e.g., CAR T cellor CAR-expressing NK cell) (e.g., more than one administration of theCAR-expressing cell (e.g., CAR T cell or CAR-expressing NK cell) perweek) is administered to the subject. In another embodiment, the subject(e.g., human subject) receives more than one cycle of CAR-expressingcell (e.g., CAR T cell or CAR-expressing NK cell), and the time betweeneach cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In oneembodiment, the CAR-expressing cell (e.g., CAR T cell or CAR-expressingNK cell) are administered every other day for 3 administrations perweek. In one embodiment, the CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) of the invention are administered for at leasttwo, three, four, five, six, seven, eight or more weeks.

In one aspect, CD123 CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) are generated using lentiviral viral vectors,such as lentivirus. CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) generated that way will have stable CARexpression.

In one aspect, CAR-expressing cells, e.g., CARTs or CAR-expressing NKcells, are generated using a viral vector such as a gammaretroviralvector, e.g., a gammaretroviral vector described herein. CAR-expressingcells, e.g., CARTs or CAR-expressing NK cells, generated using thesevectors can have stable CAR expression.

In one aspect, the CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) transiently express CAR vectors for 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transientexpression of CARs can be effected by RNA CAR vector delivery. In oneaspect, the CAR RNA is transduced into the cell (e.g., T cell or NKcell) by electroporation.

A potential issue that can arise in patients being treated usingtransiently expressing CAR cell (e.g., CAR T cell or CAR-expressing NKcell) (particularly with murine scFv bearing CARs) is anaphylaxis aftermultiple treatments.

Without being bound by this theory, it is believed that such ananaphylactic response might be caused by a patient developing humoralanti-CAR response, i.e., anti-CAR antibodies having an anti-IgE isotype.It is thought that a patient's antibody producing cells undergo a classswitch from IgG isotype (that does not cause anaphylaxis) to IgE isotypewhen there is a ten to fourteen day break in exposure to antigen.

If a patient is at high risk of generating an anti-CAR antibody responseduring the course of transient CAR therapy (such as those generated byRNA transductions), CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) infusion breaks should not last more than ten tofourteen days.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples specifically point out various aspects of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1 Human CAR Constructs

Fully human anti-CD123 single chain variable fragments were isolated.Anti-CD123 ScFvs were cloned into lentiviral CAR expression vectors withthe CD3zeta chain and the 4-1BB costimulatory molecule. CAR-containingplasmids (Table 1 provided in the Detailed Description) were amplifiedby bacterial transformation in STBL3 cells, followed by Maxiprep usingendotoxin-free Qiagen Plasmid Maki kit. Lentiviral supernatant wasproduced in 293T cells using standard techniques.

The order in which the VL and VH domains appear in the scFv was varied(i.e., VL-VH, or VH-VL orientation), and where either three or fourcopies of the “G4S” (SEQ ID NO:25) subunit, in which each subunitcomprises the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S)₃ (SEQ ID NO:28)or (G4S)₄(SEQ ID NO:27)), connect the variable domains to create theentirety of the scFv domain, as shown in Table 2 (provided in theDetailed Description).

Sequences of CAR constructs and their domain sequences are listed in theDetailed Description. Analysis of the human CAR constructs was conductedas described in, e.g. Examples 2, 3, and 6.

Example 2 Analysis and In Vitro Activity of Human scFv Bearing CARTs

Human anti-CD123 CAR constructs were evaluated for activity using aJurkat cell line containing the luciferase reporter driven by the NFATpromoter (termed JNL cells). CD123 CAR activity was measured for fourhuman CAR constructs described herein (CD123 CAR1-4) and murine CD123CAR constructs 1172 and 1176. The amino acid sequence for the 1172 and1176 constructs are provided below, and are further described andcharacterized in PCT/US2014/017328.

1172 construct: CD123 4-1BBCD3z-CAR  (amino acid sequence)(SEQ ID NO: 707) MALPVTALLLPLALLLHAARPGSDIVLTQSPASLAVSLGQRATISCRASESVDNYGNTFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPPTFGAGTKLELKGGGGSGGGGSSGGGSQIQLVQSGPELKKPGETVKISCKASGYIFTNYGMNWVKQAPGKSFKWMGWINTYTGESTYSADFKGRFAFSLETSASTAYLHINDLKNEDTATYFCARSGGYDPMDYWGQGTSVTVSSASSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR1176 construct: CD123 4-1BBCD3z-CAR (26292)  (amino acid sequence)(SEQ ID NO: 708) MALPVTALLLPLALLLHAARPGSDVQITQSPSYLAASPGETITINCRASKSISKDLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNKYPYTFGGGTKLEIKGGGGSGGGGSSGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPDQGLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYWGQGTTLTVSSASSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CAR activity was measured as activation of this NFAT-driven reporter.Lentiviral supernatants containing the CART constructs were added to JNLcells for transduction. 4-6 days after transduction, JNL cells wereeither evaluated for CAR expression by FACS as described below or mixedwith target-positive (MOLM3, K562 cells engineered to express CD123(CD123-K562)) or target-negative (K562) cell lines at an effector (JNL)to target cell line (E:T) ratio of 3:1 to trigger activation (FIG. 1).After 20 hours of co-incubation, luciferase signal was measured usingthe Bright-Glo™ Luciferase Assay on the EnVision instrument as shown inFIGS. 2A-2C.

Optimal anti-CD123 CAR constructs were selected based on the quantityand quality of the effector T cell responses of CD123 CAR transduced Tcells (“CART-CD123” or “CART-CD123 T cells”) in response to CD123expressing (“CD123+”) targets. Effector T cell responses include, butare not limited to, cellular expansion, proliferation, doubling,cytokine production and target cell killing or cytolytic activity(degranulation).

Generation of CART-CD123

The human scFv encoding lentiviral transfer vectors were used to producethe genomic material packaged into the VSVg pseudotyped lentiviralparticles. Lentiviral transfer vector DNA was mixed with the threepackaging components of VSVg, gag/pol and rev in combination withlipofectamine reagent to transfect them together in to Lenti-X 293Tcells (Clontech).

After 30 hours, the media was collected, filtered and stored at −80C.The therapeutic CART-CD123 were generated by starting with the bloodfrom a normal apheresed donor whose naïve T cells were obtained bynegative selection for T cells, CD4+ and CD8+ lymphocytes. These cellswere activated by CD3×28 beads (Dynabeads® Human T-Expander CD3/CD28,Invitrogen) at a ratio of 1:3 in RPMI 1640, 10% heat-inactivated fetalcalf serum (FCS), 2 mM L-glutamine, 1× Penicillin/Streptomycin, 100 μMnon-essential amino acids, 1 mM NaPyruvate, 10 mM Hepes, and 55 μM2-mercaptoethanol at 37° C., 5% CO₂ T cells were cultured at 1×10⁶ Tcells in 0.5 mL medium per well of a 24-well plate. After 24 hours, theT cells was blasting and 0.5 mL of viral supernatant was added. The Tcells then began to divide in a logarithmic growth pattern, which wasmonitored by measuring the cell counts per mL, and T cells were dilutedin fresh medium every two days. As the T cells began to rest down afterapproximately 10 days, the logarithmic growth waned. The combination ofslowing growth rate and T cell size approaching ˜300 fl determined thestate for T cells to be cryopreserved for later analysis.

Before cryopreserving, percentage of cells transduced (expressing theanti-CD123 CAR on the cell surface) and their relative fluorescenceintensity of expression were determined by flow cytometric analysis on aBD LSRFortessa or BD-FACSCanto using Protein L as a detection reagent.Gating histogram plots of relative fluorescent intensity from that FACSfor signal above unstained cells demonstrated the percentage oftransduced T cells. Transduction resulted in a range of CART positivecells from 12-42% as shown in FIGS. 2A-2C.

Evaluating Cytolytic Activity of CART-CD123 Redirected T Cells.

To evaluate the functional abilities of CART-CD123 T cells to killtarget expressing cells, the cells were thawed and allowed to recoverovernight.

T cell killing was directed towards CD123-expressing MOLM13 acutemyelogenous leukemia cell lines stably expressing luciferase.Untransduced T cells were used to determine non-specific backgroundkilling levels. The cytolytic activities of CART-CD123 were measured asa titration of effector:target cell ratios of 4:1 and 2-fold downwarddilutions of T cells where effectors were defined as T cells expressingthe anti-CD123 chimeric receptor. Assays were initiated by mixing anappropriate number of T cells with a constant number of targets cells.After 20 hours luciferase signal was measured using the Bright-Glo™Luciferase Assay on the EnVision instrument. As the proportion ofCD123-CART-expressing cells to untransfduced T cells was increased,killing of CD123 cells was similarly increased. The data presentedherein suggest that those cells expressing CD123 are destroyed only byCD123-CART-expressing cells and not by untransduced T cells. FIG. 3.

Cytokine Production

For measuring cytokine production of CD123 CART cells, cells expressingCD123-2, CD123-3, or CD123-4, were thawed and allowed to recoverovernight. T cells expressing an isotype control (referred to as“isotype”) were used as a non-specific control for background T celleffects. The T cells were directed towards MOLM13, PL21, or U87 cells(referred to as the target cells). The assay tested an effector:targetratio of 1.25:1 or 20:1 as noted where effectors were defined as T cellsexpressing the CD123 CAR. The assay was run 24 hours after mixing of thecells, when the media is removed for analysis of cytokines IL-2 (FIG.24A), IFN-gamma (FIG. 24B), and TNF-alpha (FIG. 24C) using the CBA-Flexkit for human cytokine detection. When CD123 CAR-expressing T cells werecultured with cancer cells expressing CD123, all CD123-CARTs producedcytokines in response to target-expressing cells.

Example 3 CART123 in AML

T Cell Transduction

Human anti-human CD123 clones NVS 2 (expressing CAR123-2), NVS 3(expressing CAR123-3), NVS 4 (expressing CAR123-4) were selected forfurther study. These clones were all cross-reactive against cynomolgusCD123. Their activity was compared against mouse clones 1172 and 1176,comprising the VH and VL domains in a light-to-heavy orientation with aCD8 hinge domain, CD8 transmembrane domain, and 41BB-costimulatorydomain. 1176 is also cross-reactive against cynomolgus CD123. 1172 isnot.

Plasmids were transformed into competent cells, grown in 500 cc broth,isolated by maxiprep, and transduced using standard methods into 293Tcells. Lentiviral supernatant was collected at 24 and 48 hours,concentrated using ultracentrifugation, and frozen.

The lentivirus was titered on SupT1 cells and the appropriate amount ofvirus was determined for a transduction of primary T cells at a MOI of3. Primary normal donor CD4+CD8 cells were stimulated usinganti-CD3/CD28 beads (Dynal, Invitrogen) and interleukin-2 100 U/ml for 6days, followed by debeading and were and frozen once. The T cellcellular volume decreased to <300 fL (after approx 10-12 days).

T cell transduction efficiency was virtually 100% for all clones (FIGS.5A and 5B). CD4:CD8 ratios were approximately 1:1 in the NVS clones, and3:2 in the 1172 and 1176 clones (FIG. 6).

Degranulation

To assess degranulation, CART cells (NVS 2-4, 1172 and 1176 clones) werethawed, rested overnight at a concentration of 2e⁶ cells/ml in T cellmedia. Cells were then counted and resuspended at 1e⁶ cells/ml thefollowing day. Tumor target cells (TCM, PMA/iono, MOLM14 or JURKAT) wereresuspended at 5e⁶ cells/ml. Cells were plated at a ratio of 1e⁵ T cell:5e⁵ tumor cell in 48 well plates and incubated for 2 hours in thepresence of anti-CD107a PECy7, anti-CD49d purified, and anti-CD28purified antibodies. Cells were then harvested, stained with anti-CD3APC and acquired using BD LSR Fortessa (FIG. 7).

T cell degranulation is indicated in the upper right quadrant of eachplot of FIG. 7. The results presented herein demonstrate similar T cellrecognition of CD123+ targets, manifested by similar degranulationduring a 2-hr in vitro assay. C1176 had inferior degranulation of 65%compared with approximately 80% in the other clones.

Cytotoxicity

To assess cytotoxicity, CART cells (NVS 2-4, 1172 and 1176 clones) werethawed and rested overnight at 2e⁶ cells/ml in T cell media. Cells werecounted and resuspended at 1e⁶ cells/ml the following day. Tumor targetcells (MOLM14) were resuspended at 1e⁶ cells/ml. Cells were plated in ablack, flat-bottom 96 well plate at decreasing E:T ratios as indicated(FIG. 8), in duplicate. After 20 hours of incubation, luciferin wasadded and the plate was imaged to determine photon flux as a measure ofresidual live cells. Killing of MOLM14 cells was equivalent between allclones at most effector:target ratios at 20 hours.

In Vivo Mouse Model

NSG mice were injected iv with 1e⁶ luciferase expressing MOLM14 cells onD0. On D6 mice were imaged (IVIS Spectrum) for tumor burden andrandomized into treatment groups. Mice with the lowest tumor burden wereassigned to the control group (untransduced T cells, UTD). CART cells(NVS 2-4, 1172 and 1176 clones) or control T cells (1e6) were injectedi.v. on D7. Data from imaging on performed D13 is shown in FIG. 9. Sixdays after injection, all anti-CD123 constructs provided equalanti-tumor effect, consistent with in vitro data.

Example 4 Humanized CAR Constructs

Humanized anti-CD123 single chain variable fragments (scFv) based onmurine 1176 which is cross-reactive against cynomolgus CD123 weregenerated and cloned into a lentiviral expression vector with theintracellular CD3zeta chain and the intracellular co-stimulatory domainof 4-1BB and given the names depicted in Table 5 (provided in theDetailed Description).

The order in which the VL and VH domains appear in the scFv was varied(i.e., VL-VH, or VH-VL orientation), and where either three or fourcopies of the “G4S” (SEQ ID NO:25) subunit, in which each subunitcomprises the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S)₃ (SEQ ID NO:28)or (G4S)₄(SEQ ID NO:27)), connect the variable domains to create theentirety of the scFv domain, as shown in Table 6 (provided in theDetailed Description).

The sequences of the humanized scFv fragments (SEQ ID NOs: 184-215) CARsare provided herein in Table 6. These clones all contained a Q/K residuechange in the signal domain of the co-stimulatory domain derived fromCD3zeta chain. The CAR scFv fragments were then cloned into lentiviralvectors to create a full length CAR construct in a single coding frame,and using the EF1 alpha promoter for expression (SEQ ID NO: 11).

Example 5 CART123 in Hodgkin's Lymphoma

CD123 is known to be expressed in around 50% of classical Hodgkin'sLymphoma (HL) by flow cytometry. Exogenous administration of IL3 toHodgkin's Lymphoma cell line cultures is known to promote growth and isable to partially rescue from cells from apoptosis by serum deprivation.Inhibition of CD123 (IL-3Ra) by SL-401 is known to to reduced viabilityof CD123++ HL cell lines (HDLM-2, L428).

8 patients with HL were analyzed for CD123 expression buyimmunohistochemistry. As shown in Table 15, 4/8 samples showedpositivity in Reed-Sternberg cells and/or in immune cell of themicroenvironment.

TABLE 15 Target expression by IHC in 8 HL specimens UPN CD30 CD12311-978 + − 11-33317 + − 13-4203 + − (histiocytes+) 11-20838 + +11-4623 + − 11-17638 D/E + + (subset) 13-14768 + +

Flow cytometry showed expression of CD30 and CD123 in a well-knownHodgkin's Lymphoma cell line HDLM-2 (FIG. 10). B-cell markers CD19 andCD20 were absent.

The experiments below use a pELNS anti-CD123-41BB-CD3 (CAR123) plasmidDNA with light-to-heavy or heavy-to-light chain orientations of themouse anti-human CD123 scFv. T cells were obtained from a normal donor(ND052, Human Immunology Core), expanded using CD3/CD28 magnetic beads(Invitrogen) and transduced on day 2 with CAR123 lentivirus. At day 6CD123 was detected via detecting dsRed, surrogate marker of CD123 by T2Acoexpression. dsRed was detected via flow cytometry (FIG. 11A-B).

Q-PCR was then performed on CD123 expression in 4 Hodgkin's Lymphomacell lines (HDLM2, KMH2, L428, SUPHD1), CD123+ MOLM14 (positive control)and A375 (negative control). CD123 was expressed in all Hodgkin'sLymphoma cell lines (FIG. 12). GUSB was used as a housekeeping gene. TheCt threshold was 40.

Role of IL-3 in Promoting HL Cell Growth

As CD123 is the IL3 receptor a chain and IL-3 has important roles inhematopoietic growth and differentiation, it was investigated whetherIL3 signaling is important in the growth of Hodgkin's Lymphoma celllines. NOD-SCID-γ-chain KO mice that overexpress human cytokinesincluding IL-3 (NSG-S mice) were engrafted with theluciferase-expressing HDLM-2 cell line. After i.v. injection, theneoplastic cells progressively formed disseminated soft tissue masses.Serial injections of a neutralizing anti-IL3 antibody slowed the growthof tumor. The results presented herein suggest that CD123 may be aparticularly relevant target in Hodgkin's Lymphoma. Tumor burden isshown by BLI (FIG. 13).

CD107a Degranulation Assay

Untransducted T cells (UTD) or CART123 were incubated with HDLM-2(CD123+ HL cell line) for 4 hours at a ratio of 5 target cells to 1 Tcells in the presence of anti-CD28, anti-CD49d antibodies and monensin.Anti-CD30 CAR T cells were used as additional controls.

CART123 but not UTD showed increase CD107a degranulation as detected byflow cytometry (FIGS. 14 and 15). Moreover CART123 showed significantincrease in intra-cytoplasmatic cytokine production (IL-2, TNF) (FIG.16).

CART123 Kill HDLM-2 Cells

A luciferase based 24 hr killing assay was performed. T cells wereco-incubated with luciferase+ HDML-2 cells at different rations(0.3:1-10:1). CART123 but not UTD showed a dose dependent killing asshown by decrease in bioluminesce emission. Interestingly, CD30-specificCAR T cells showed minimal activity (FIG. 17).

CART123 Proliferate in the Presence of HL Cell Lines

T cells were incubated with HL cell lines (CD123+) or controls (JurkatCD123-, MOLM-14 CD123+) or PMA/Ionomycin (positive control) or cellmedia (negative control) in a 5-day T cell proliferation assay. CART123and CART30 cells but not UTD showed robust proliferation whenco-cultured with HL cell lines (FIG. 18)

Supernatant from the proliferation assay was collected after 72 hrs andthe presence of 30 cytokines was analyzed by Luminex assay. CART123showed robust production of multiple cytokines (IFNg, IL-2, TNFa andMIP1b luminex MFI are shown in the FIGS. 19A-19D, respectively).

In Vivo Efficacy of CART123 Against a Hodgkin's Lymphoma Cell Line(HDLM-2)

To confirm the in vitro data presented herein, an in vivo model wasdeveloped. 1 million luciferase+HDLM-2 cells were injected i.v. on day0. Serial bioluminescent imaging (BLI) demonstrated was then performedto observe tumor level (FIG. 20) A low level of tumor was observed onday 7, which was followed by gradual increase in tumor burden overapproximately 6 weeks, reproducing the indolent nature of the humandisease. At day 43 when the tumor burden was 20-fold higher thanbaseline, mice were treated with 1.5 million CART123 cells or control Tcells.

CART123 induced complete and durable eradication of disseminated tumorwithin 14 days, leading to 100% relapse-free and 100% overall survivalat 6 months (FIGS. 21 and 22).

Tumor elimination was associated with extensive CAR T cell expansion asdetected by flow cytometry in serial peripheral blood bleedings (FIG.23). The expanded T cells were approximately 50% CD8 and 50% CD4 cells.The T cell number contracted over time as tumor burden decreased.

Example 6 Additional Characterization of CD123-2 CAR

Additional in vitro assays were performed to evaluate primary T cellsthat were lentivirally transduced to express the CAR123-2 construct(also referred to herein as LV CAR123-2). The CAR-expressing T cells (LVCAR123-2) were compared to T cells expressing an scFv againstcytomegalovirus (hCMV) glycoprotein H (gH) (referred to herein asanti-GH), which acted as a negative control. Key in vitro assaysincluded cytokine production, proliferation, and cell killing assays.Proliferation assays were performed by incubating T cells (LV CAR123-2or anti-GH) with CD123-expressing (antigen+) cell lines, e.g., MOLM13 orK562 expressing human CD123, or CD123-negative (antigen−) control celllines, e.g., K562. The T cells were grown for 4 days. As shown in FIG.25, the CAR-expressing cells exhibited robust proliferation compared tothe negative control when cultured in the presence of CD123-expressingcells.

The ability of CAR123-2-expressing T cells to kill target cells wasmeasured as a titration of target:effector cell ratios ranging from 1:20and two-fold dilutions to 1:0.3125. The target cells that were testedwere CD123-expressing MOLM13 cell lines that stably express luciferase,PL21 cells that stably express luciferase, and U87 cells that stablyexpress luciferase. As shown in FIGS. 26A and 26B, the CD123-CARexpressing cells demonstrate targeting killing of CD123-expressingcancer cells (MOLM13 and PL21), while the negative control (cellsexpressing anti-GH) and untransduced cells (UTD) do not demonstratetargeted killing. In FIG. 26C, UTD, negative control (anti-GH) and theCD123 CAR-expressing cells did not demonstrate specific killing of U87cells (which do not express CD123).

A cytokine production assay was performed by analyzing the supernatantfrom co-cultures comprising T cells (expressing either CAR123-2 oranti-GH) with antigen positive (MOLM13) or antigen negative (U87) cells.CD123-CAR T cells showed robust cytokine production of IFN-gamma (FIG.27A) and IL-2 (FIG. 27B).

Example 7 Combination of Anti-CD123 and Anti-CD19 CAR T Cells for theTreatment and Prevention of Antigen-Loss Relapse

Chemo-refractory or relapsing (r/r) B-cell acute lymphoblastic leukemia(B-ALL) is associated with a poor prognosis but, as demonstratedrecently, remains exquisitely sensitive to the immune system. Inparticular, anti-CD19 chimeric antigen receptor T cells (CART19, CTL019)and bi-specific anti-CD19/CD3 antibodies (blinatumomab) generateunprecedented complete response rates of 45-90% in this patientpopulation. Both approaches re-direct autologous T cells to recognizeCD19-expressing cells. Blinatumomab uses a continuous long-term infusionof a bispecific construct that combines an anti-CD19 single chainvariable fragment (scFv) with an anti-CD3 scFv; in the case of CART19 Tcells are genetically modified to express an anti-CD19 scFv fused to theT cell receptor signaling with built-in co-stimulatory domains. A recentstudy showed that 90% of patients with r/r B-ALL treated with CTL019reach complete remission (CR) with an overall survival (OS) of 78% at 6months. Encouraging results with CART19 were also obtained in patientswith other B-cell neoplasms, such as chronic lymphocytic leukemia andnon-Hodgkin lymphoma.

However, a subset of patients treated with CART19 or blinatumomabdevelops relapse and a significant portion of these relapses arecharacterized by the loss of CD19. In B-ALL, CD19-negative relapses havebeen reported in 10-20% of patients following CART19 or blinatumomabtherapies and it has not been described in the setting of othertreatments; overall about 30% of relapses after blinatumomab and up to50% after CART19 are CD19-negative. CD19 is a prototypic B-cell markerthat is expressed from the very earliest stages of B cell development tothe mature B-cell. CD19 plays an important role in B cell biology asCD19-deficient B cells exhibit selective growth disadvantage. Thus theabsence of CD19 is a very unusual finding in B-ALL and it is has beenreported in only rare patients prior to the era of potent CD19-directedimmunotherapies. The possible mechanism of antigen loss is currentlyunder investigation and is most likely caused by selective pressure onleukemia sub-clones by these powerful anti-CD19 agents. Because of therecent approval by the FDA of blinatumomab and the breakthrough statusaccorded to CTL019, it is likely that increasing numbers of patientswith r/r B-ALL will be treated with these agents. Hence, novel effectivestrategies are needed in order to be able to treat those patients thatwill relapse with CD19-negative blasts after CART19 or blinatumomab.Ideally a new approach would not only treat patients with activeantigen-loss relapse but if employed upfront could potentially preventtheir occurrence.

The interleukin-3 receptor alpha (or CD123) is involved in hematopoiesisand has been shown to be expressed in several hematologic neoplasms,including acute myeloid leukemia (AML), acute lymphoid leukemia (ALL),plasmacytoid dendritic cell neoplasm, hairy cell leukemia, and Hodgkinlymphoma. Unlike lineage-associated surface antigens such as CD33(myeloid) or CD19 (B-lymphoid), CD123 is hierarchically expressed onhematopoietic progenitor cells and in AML CD123 is expressed on leukemicstem cells that are involved in resistance to chemotherapy and relapseafter initial treatment. Due to these characteristics, CD123 hasgenerated great interest for targeted therapy, and multiple agents arebeing developed such as the IL3-diphtheria toxin fusion protein (SL-401,DT388IL3), naked anti-CD123 monoclonal antibodies (CSL-360, CSL-362),antibody-drug conjugates, bi-specific antibodies or CD3Fv-IL3 fusionconstructs, and more recently, anti-CD123 chimeric antigen receptor Tcells. Some of these approaches are currently being validated inclinical trials and many more will be tested in the clinic in the nextfew years. Targeting CD123 with chimeric antigen receptor T cells(CART123) can lead to deep and long-term responses in human primary AMLxenografts and can establish an anti-leukemia T cell memory. Here, CD123is expressed in CD19-negative B-ALL relapses occurring afterCD19-directed therapies and that CAR-123 T cells combined with CART19(CTL019) is an effective therapy for the treatment and for theprevention of antigen-loss relapses in B-ALL xenografts.

Materials and Methods

Cell lines and primary samples. Cell lines were originally obtained fromATCC (Manassas, Va.) (K-562) or DSMZ (Braunschweig, Germany) (MOLM-14and NALM-6). All cell lines were tested for the presence of mycoplasmacontamination (MycoAlert™ Mycoplasma Detection Kit, LT07-318, Lonza,Basel, Switzerland). For some experiments, cell lines were transducedwith firefly luciferase/eGFP and then sorted to obtain a >99% positivepopulation. The luciferase positive K-562 cell line was also transducedwith truncated CD19 or truncated CD123 to obtain cell lines expressingneither of them, only CD19 or only CD123. MOLM-14 and K562 were used ascontrols as indicated in the relevant figures. The cell lines weremaintained in culture with RPMI media 1640 (Gibco, 11875-085,LifeTechnologies, Grand Island, N.Y.) supplemented with 10% fetal bovineserum (FBS, Gemini, 100-106, West Sacramento, Calif.), and 50 UI/mlpenicillin/streptomycin (Gibco, LifeTechnologies, 15070-063).De-identified primary human ALL bone marrow (BM) and peripheral blood(PB) specimens were obtained from the clinical practices of Universityof Pennsylvania/Children's Hospital of Philadelphia under anInstitutional Review Board (IRB)-protocol, purchased from the Stem Cellsand Xenograft Core of the University of Pennsylvania or from researchsamples of the current CTL019 clinical trials (Translation andCorrelative Study Laboratory, at the University of Pennsylvania). Forall functional studies, primary cells were thawed at least 12 hoursbefore experiment and rested at 37° C.

In vivo expansion of primary B-ALL blasts. Methods disclosed in D. M.Barrett, A. E. Seif, C. Carpenito, D. T. Teachey, J. D. Fish, C. H.June, S. A. Grupp, G. S. Reid, Noninvasive bioluminescent imaging ofprimary patient acute lymphoblastic leukemia: a strategy for preclinicalmodeling. Blood 118, e112-117 (2011).

Fluorescence in situ hybridization (FISH) and immunohistochemistry. TheFISH analysis and immunohistochemistry were performed according to thestandard method and as described. (M. A. Belaud-Rotureau, M. Parrens, P.Dubus, J. C. Garroste, A. de Mascarel, J. P. Merlio, A comparativeanalysis of FISH, RT-PCR, PCR, and immunohistochemistry for thediagnosis of mantle cell lymphomas. Modern pathology: an officialjournal of the United States and Canadian Academy of Pathology, Inc 15,517-525 (2002)). The FISH analysis was performed according to thestandard method. In brief, harvested ALL cells were suspended infixative (acetic acid and methanol), deposited on the slides, and leftto dry. The dual color gene fusion probe BCR/ABL (Abbott Molecular), wasapplied in the hybridization buffer solution. The slides werecover-slipped, sealed, and left inside the HYBrite chamber at 37 C for 6hr. After removal of the sealant and the coverslip, the slides werewashed twice, blotted, dried, and counterstained with DAPI. The slideswere examined under fluorescent microscope, with a minimum of 200 nucleievaluated in each specimen.

Generation of CAR constructs and CAR T cells. The murine anti-CD19chimeric-antigen receptor (CD8 hinge, 4-1BB co-stimulatory domain andCD3 zeta signaling domain) was generated as previously described.(Milone, et al., Molecular therapy: the journal of the American Societyof Gene Therapy 17, 1453-1464 (2009) and Imai, et al., Leukemia 18,676-684 (2004)). This is the same construct currently used in the CTL019clinical trials at the University of Pennsylvania. For CAR123 scFvanti-CD123 (1172 construct (SEQ ID NO: 707, and as described inPCT/US2014/017328) was used and the same backbone construct of CAR19.Production of CAR-expressing T cells was performed as previouslydescribed. (Gill, et al., Blood 123, 2343-2354 (2014)). Normal donor CD4and CD8 T cells or PB mononuclear cells (PBMC) were obtained from theHuman Immunology Core of the University of Pennsylvania. T cells wereplated at 1×10⁶/ml with a CD4:CD8 ratio of 1:1 and expanded in X-vivo 15media (Lonza, 04-418Q), supplemented with human AB serum 5% (Gemini,100-512), penicillin/streptomycin (Gibco, 15070063) and Glutamax (Gibco,35050061) using anti-CD3/CD28 Dynabeads (Life Technologies, 11161D)added on the day 1 of culture and removed on day 6. T cells weretransduced with lentivirus on day 2. T cells were expanded in culturefor 8-15 days and harvested when the median cell volume was below 300fl. T cells were then cryopreserved in FBS with 10% DMSO for futureexperiments. Prior to all experiments, T cells were thawed and restedovernight at 37° C.

Multiparametric flow cytometry. Flow cytometry was performed aspreviously described (Kenderian, et al., Leukemia, (2015)). Anti-humanantibodies were purchased from Biolegend, eBioscience, or BectonDickinson. Cells were isolated from in vitro culture or from animals,washed once in PBS supplemented with 2% fetal calf serum, and stainedfor 15 minutes at room temperature. For cell number quantitation,Countbright (Invitrogen) beads were used according to the manufacturer'sinstructions. In all analyses, the population of interest was gatedbased on forward vs. side scatter characteristics followed by singletgating, and live cells were gated using Live Dead Fixable Aqua(Invitrogen). Time gating was included for quality control. Surfaceexpression of CAR19 was detected as previously described, using ananti-idiotype antibody. Detection of CAR123 was performed usinggoat-anti-mouse antibody (Jackson Laboratories) or CD123-Fc/His (SinoBiologicals) and anti-His-APC (R&D) or PE (AbCam). Flow cytometry wasperformed on a four-laser Fortessa-LSR II cytometer (Becton-Dickinson)and analyzed with FlowJo X 10.0.7r2 (Tree Star).

In vitro T-cell effector function assays. Degranulation, CFSEproliferation, cytotoxicity assays and cytokine measurements wereperformed as previously described. (Gill, et al., Blood 123, 2343-2354(2014) and Kalos, et al., Science translational medicine 3, 95ra73(2011)).

Degranulation assay. Briefly, T cells were incubated with target cellsat a 1:5 ratio in T cell media. Anti-CD107a-PECY7 (Biolegend), anti-CD28(BD Biosciences), anti-CD49d (BD Biosciences) antibodies and monensin(BD Biosciences) were added to the co-culture. After 4 hours, cells wereharvested and stained for CAR expression, CD3, CD8 and Live Dead aquastaining (Invitrogen). Cells were fixed and permeabilized (InvitrogenFix/Perm buffers) and intracellular staining was then performed todetect multiple cytokines (IFN, TNFα, IL-2, GM-CSF, MIP1β).

Proliferation assay. T cells were washed and resuspended at 1×107/ml in100 ul of PBS and stained with 100 ul of CFSE 2.5 uM (Invitrogen) for 5minutes at 37° C. The reaction was then quenched with cold media, andcells were washed three times. Targets were irradiated at a dose of 100Gy. T cells were incubated at a 1:1 ratio with irradiated target cellsfor 120 hours, adding media at 24 hours. Cells were then harvested,stained for CD3, CAR and Live Dead aqua (Invitrogen), and Countbrightbeads (Invitrogen) were added prior to flow cytometric analysis forabsolute quantification.

Cytotoxicity assays. Luciferase/eGFP+ cell lines were used forcytotoxicity assay as previously described. In brief, targets wereincubated at the indicated ratios with effector T cells for 24 hours.Killing was calculated by bioluminescence imaging on a Xenogen IVIS-200Spectrum camera.

Cytokine measurements. Effector and target cells were co-incubated at a1:1 ratio in T cell media for 24. Supernatant was harvested and analyzedby 30-plex Luminex array (Luminex Corp, FLEXMAP 3D) according to themanufacturer's protocol (Invitrogen).

Animal experiments. In vivo experiments were performed as previouslydescribed. (Kenderian, et al., Leukemia, (2015)). Schemas of theutilized xenograft models are discussed in detailed in the relevantfigures, result. NOD-SCID-γ chain−/− (NSG) originally obtained fromJackson Laboratories were purchased from the Stem Cell and XenograftCore of the University of Pennsylvania. All experiments were performedaccording a protocol (#803230) approved by the Institutional Animal Careand Use Committee (IACUC) that adheres to the NIH Guide for the Care andUse of Laboratory Animals. Cells (leukemia cell lines or T cells) wereinjected in 200 ul of PBS at the indicated concentration into the tailveins of mice. Bioluminescent imaging was performed using a XenogenIVIS-200 Spectrum camera and analyzed with LivingImage software v. 4.3.1(Caliper LifeSciencies). Animals were euthanized at the end of theexperiment or when they met pre-specified endpoints according to theIACUC protocols.

Multiphoton microscopy. Mice were anaesthetized and maintained at coretemperature of 37° C. Bone marrow was imaged after removing the scalpand immobilizing the skull. Imaging was performed using a Leica SP52-photon microscope system (Leica Microsystems) equipped with apicosecond laser (Coherent). Each imaging acquisition lasted 20 minfollowed by an assessment of mouse sedation. CellTrace Violet, GFP, andCellTrace Orange (or TRITC) were excited using laser light of 850 nm.Images were obtained using a 20× water-dipping lens. The resultingimages were analyzed with Volocity software (PerkinElmer).

Statistical analysis. All statistics were performed as indicated usingGraphPad Prism 6 for Windows, version 6.04 (La Jolla, Calif.). Student'st-test was used to compare two groups; in analysis where multiple groupswere compared, one-way analysis of variance (ANOVA) was performed withHolm-Sida correction for multiple comparisons. When multiple groups atmultiple time points/ratios were compared, the Student's t-test or ANOVAfor each time points/ratios was used. Survival curves were comparedusing the log-rank test. In the figures asterisks are used to representp-values (*=<0.05, **=<0.01, ***=<0.001, ****=<0.0001) and “ns” means“not significant” (p>0.05). Further details of the statistics for eachexperiment are listed in figure legends.

Results

CD123 is Expressed in B-ALL, in the Leukemia Stem Cells and inCD19-Negative Relapses

In order to evaluate the expression of CD123 in B-cell acutelymphoblastic leukemia, 42 samples from adult and pediatric ALL patientswere analyzed, including 14 subjects enrolled in our current CTL019clinical trials. As shown in FIGS. 31A, 31B and 38A, CD123 is highly andhomogeneously expressed on the surface of most ALL blasts, representingan ideal candidate for targeted therapy. Moreover, CD123 is also foundto be expressed in the putative leukemia stem cells (LSC), identified asCD34+ CD38− (FIG. 31C). Small subsets of CD19-negative blasts can beidentified in some B-ALL patients and these cells could contribute toantigen-loss relapses, if they contained cells with a malignantphenotype. In order to evaluate the presence of disease in CD19-negativesubsets, CD19− CD123+ cells from a Philadelphia chromosome positiveB-ALL bulk population were sorted (CD45dim, gating strategy shown FIG.38B). It was found that these cells were clonal for the BCR-ABLtranslocation, albeit at a lower frequency than the CD19+ blasts (FIG.31D). This finding indicates that targeting CD19 alone could, in somecases, lead to a subclonal relapse derived from CD19−CD123+ cells.Furthermore, this finding suggests that targeting CD123 could lead todeeper responses through the elimination of the LSC and possiblyCD19-neg leukemia clones.

Finally the expression of CD123 was also evaluated in the samples ofB-ALL patients relapsing after CTL019 with loss of CD19. Importantly, incontrast to the complete loss of CD19, the majority of patientsmaintained CD123 expression at relapse (FIGS. 31E, 31F and 38C). Thesefindings indicate that CD123 represents an ideal marker to targetCD19-neg ALL blasts occurring after CART19 or blinatumomab.

Anti-CD123 Chimeric Antigen Receptor T Cells are Active Against HumanB-ALL In Vitro and In Vivo

Anti-CD123 chimeric antigen receptor T cells (CART123) were generated(37) that were lentivirally transduced and expanded with anti-CD3/CD28magnetic beads. The in vitro and in vivo activity of CART123 againstB-acute lymphoblastic leukemia were evaluated as described herein.

The B-ALL cell line NALM-6 that is CD19++ and CD123+ (FIGS. 32A and 39A)and primary B-ALL samples were used. A head-to-head in vitro comparisonbetween CART123 and CART19 revealed similar rates of CD107adegranulation when T cells were co-cultured with NALM-6 or primary ALL(FIG. 32B). CART123 were also able to kill NALM-6 cells with similarefficacy as CART19 in a dose-dependent manner (FIG. 32C). At morelong-term experiments, CART123 proliferated (FIGS. 32D and 32E) andproduced multiple cytokines (FIG. 32F) when co-cultured with NALM-6 orprimary ALL for 3-5 days. These results indicate that CART123 exhibitequivalent potency to CART-19 against multiple B-ALL targets.

In order to confirm these data in an in vivo model, a primary ALL modelwas utilized. In this model, primary blasts obtained from B-ALL patientswere passaged in NOD-SCID-γ chain knock-out (NSG) mice and transducedwith a reporter construct containing eGFP and click beetle luciferase(GFP/Luc). NSG mice were injected with GFP/Luc+ primary ALL blasts i.v.(JH331, CD19+, CD123+, add phenotype) and after engraftment, mice wererandomized to receive CART19, CART123 or control untransduced T cells(UTD). Mice treated with control T cells succumbed quickly to disease,while mice treated with either CART19 or CART123 showed tumoreradication and long term survival (FIGS. 33A and 33B). CAR123 T cellssignificantly expanded in the peripheral blood (PB) of the mice comparedto control T cells and expressed high levels of CAR123 (FIG. 33C). Theanti-leukemia activity of CART123 was specific and based on therecognition of CD123 in the surface of the blasts as when we engraftedmice with a CD123−CD19+ leukemia (AV576), only CART19 show anti-leukemiaactivity, while CART123 had no effect as compared to controls UTD (FIGS.39B and 39C).

In order to detect a possible correlation of CART123 dose and anti-tumoractivity, an in vivo model of high leukemia burden bearing mice (usingthe NALM-6 cell line) was developed. In this model standard doses ofCART123 (2 million CAR+ cells) are not able to clear the tumor. Thesemice were injected with different doses of CART123 (1.25, 2 and 5million CAR+ cells) and observed a dose-related anti-leukemia activity(FIG. 39D).

CART123 but not CART19 are Highly Active in a Novel Preclinical Model ofAntigen-loss Relapse

In order to test new strategies to target CD19-negative relapses a novelin vivo model of antigen-loss relapse was developed. B cell blastsobtained from a patient (CHP101) enrolled in one of our CTL019 clinicaltrials were collected at baseline (before CTL019 therapy), when thedisease was CD19++ and CD123+, and at relapse after CTL019 when thepatient developed a CD19-negative disease (CD123 still expressed, FIG.40A). Blasts were then expanded in NSG mice and transduced withclick-beetle green luciferase (CBG) for baseline disease (CD19+) orclick-beetle red luciferase (CBR) for relapse (CD19−) (see Methodssection). Importantly, during in vivo expansion, the blasts retainedmarkers of B cell identity other than CD19 (data not shown). In a firstexperiment NSG mice were engrafted with either the baseline diseaseCD19+ (CBG, green) or the CD19-neg (CBR, red) leukemia. Both groups wererandomized to receive CART19 or control T cells (UTD) (FIG. 34A). Asshown in FIG. 34B, in both groups mice treated with UTD showed rapidprogression of both the baseline and relapse disease, independently bythe expression of CD19. Conversely, in the group of mice treated withCART19, only mice engrafted with the baseline disease (CD19-positive)responded to CART19 treatment while mice engrafted with the relapseddisease (CD19-negative) showed refractoriness as expected. This was alsoreproduced in vitro in a CD107a degranulation assay (FIG. 40B). In orderto simulate in vivo the presence of different clones expressing CD19 orlacking it, NSG mice were engrafted with a 1:1 mixture of baseline andrelapse disease; at day 8 mice were randomized to receive CART19 orcontrol T cells. Tumor burden was monitored with bioluminescence imagingthat could discriminate between CD19+(CBG, green)/CD19− (CBR, red)leukemia relative growth in vivo. As shown in FIG. 43C, in micereceiving UTD both CD19+ (green) and CD19− (red) leukemia present at day6 was similarly increased at day 11, while in mice treated with CART19the baseline disease (green) was completely cleared while the relapseddisease (red) showed progression.

This unique xenograft model of primary CD19-negative B-ALL and CART19failure was used to evaluate the role of CART123 in the treatment ofantigen-loss relapses. Primary CD19-negative blasts (CBR positive) wereinjected into NSG mice (FIG. 34D) and mice were randomized to receiveCART19, CART123 or control T cells. CART19 and control T cells showedcomplete lack of anti-tumor activity, while CART123 lead to completeeradication of the disease and long term survival in these mice (FIGS.34E and 34F). Indeed in a pre-clinical model of primary B-ALL refractoryto CART19, the novel CART123 are able to eradicate the disease andconfer long-term survival.

In order to understand the differential behavior of CART19 and CART123in this in vivo model at a single cell level, a series of experimentswas performed by injecting a mixture of differentially labelled CART19(CellTrace Violet, blue) and CART123 (CellTracker Orange or TRITC, red)to mice bearing CD19-positive primary blasts (GFP) or CD19-negativerelapsed blasts (GFP) and tracked their behavior using intravital2-photon microscopy of calvarial marrow approximately 24 hours afterinjection (experiment schema, FIG. 35A). These studies showed thatCART19 and CART123 trafficked to marrow spaces containing leukemia andthat CART cell recognition of cognate antigen correlate with motilityarrest. Specifically, in mice engrafted with the baseline CD19+CD123+leukemia, 62.9%+/−3.8 of CART19 and 81.1%+/−1.2 of CART123 were found tobe stalled with a rounded morphology adjacent to blasts, whereas in miceengrafted with the relapsed CD19−CD123+ leukemia, only CART123 cellsarrested next to tumor cells (CART123 80.9%+/−5.1 vs CART19 12.4%+/−2.2)(FIGS. 35B and 35C). These findings indicate that in CD19-negativerelapsed ALL only CART123 were able to establish productive synapseswith the leukemia cells (GFP) and thus reduced their motility, whereasCART19 cells continued sampling and moving in the environment withoutrecognizing the leukemia blasts.

The Combination of CART123 and CART19 is Able to Prevent CD19-NegativeRelapses

CART123 proved to be effective in the treatment of CD19-negativerelapses occurring after CD19-directed therapies in a preclinical modelof CART19 resistance. However, a combinatorial approach could treatactive CD19-positive disease while simultaneously preventingantigen-loss relapses. In order to test this hypothesis the emergingclinical problem of B-ALL with a potential for CD19-negative escape wasmodeled by injecting primary CD19− and CD19+ disease together into NSGmice. Mice were then randomized to receive control T cells (UTD), CART19or the combination of CART19 and CART123, with the same total dose of Tcells (FIG. 36A). As shown in FIG. 36B, mice treated with control Tcells had progression of both leukemia clones and CART19 showed rapidprogression mostly of the CD19-neg disease (red). On the contrary micetreated with the combination of CART123 and CART19 showed clearance ofthe disease and improved overall survival, as shown in FIG. 36C.Analysis of mice sacrificed at the end of the experiment showed noevidence of residual leukemia in the pooled CAR T cell group. Incontrast, mice with progressive disease after CART19 monotherapyretained the expected CD19 negative phenotype (FIG. 36D).

Lastly, T cells were transduced with 2 lentiviruses, one carrying CAR19and the other CAR123 in order to develop a CART able to be activated byboth CD19 and/or CD123. As shown in FIG. 37A, four differentlytransduced T cell subsets were detected: CAR19 and CAR123 doublenegative, CAR19 single positive, CAR123 single positive and doublepositive CAR19/CAR123 T cells. These four subsets were sorted and theirfunctionality and specificity was tested against K562-WT, K562 CD19+ orK562 CD123+. FIG. 37B shows the results of a CD107a degranulation assaywhere the single positive subsets respond to their specific target whileonly the double positive population is able to degranulate in thepresence of both CD19 and CD123 expressing K562. In addition,dually-stimulated CART cells exhibited more potent cytotoxicity againsta double-positive target in comparison with an equivalent number ofsingle-stimulated CART cells, suggesting a potential increment inefficacy by using a CAR that is triggered by two different antigens.(FIG. 37C)

Discussion

CD19 directed immunotherapies are changing the paradigm of treatment ofrelapsing and refractory acute lymphoblastic leukemia. Patients with apreviously dismal outcome now have a realistic potential to achieve acomplete response and long-term disease remission. However, as shownunder some circumstances for leukemia treated with other forms of potenttargeted therapy, leukemia cells are able to develop antigen-lossmutations that lead to resistance and relapse. In the case of CART19,two main patterns of relapses have been observed. Patients with earlyloss of CART19 through failure of persistence are at risk for relapse ofthe original clone; indeed, minimal residual disease analyses indicatethat between 1-6 months of sustained CART activity may be required tocompletely eradicate malignancy. In contrast, around 50% of relapsesoccur despite CART19 persistence and are characterized by the occurrenceof aCD19-negative leukemia. The latter observation implicates potentselective pressure by CART19. Notably, CD19-negative relapses have alsooccurred after blinatumomab therapy although these represent theminority of relapse occurrences after this arguably less potent therapy.(5) There are multiple potential mechanisms for the development ofCD19-negative disease. One of these is the selection and relativesurvival advantage of CD19-negative clones that were present at baselinein very low frequency, and this was initially considered the most likelyfactor leading to CD19-negative relapses. More recently othermechanisms, such the dysregulation of the splicing of CD19 have alsobeen considered important. Here it is shown for the first time that rareCD19-ve blasts in B-ALL can contain the hallmark cytogeneticabnormalities found in the more common CD19-positive leukemia blasts,confirming this as a potential mechanism of CD19-negative relapse. Weconfirmed these findings by demonstrating that CD123+ CD19-ve blasts canengraft in immunodeficient mice, indicating that CD123 may be a markerof leukemic stem cells in B-ALL as it is in AML.

The goal of this study was to define novel strategies to treat patientsrelapsing with antigen loss after CD19-directed therapies. CD123 washighly expressed in the majority of B-ALL, and in particular CD123remains expressed in those relapsing with CD19-negative disease. It wasdemonstrated the presence of clonal leukemic cells in the CD19− CD123+population indicating that targeting CD123 in combination with CART19can increase the likelihood of eradicating sub-clones that couldproliferate due to a selective advantage upon CART19 pressure. CD123 haspreviously been validated as a marker of the leukemic stem cell in AML.Here it was shown that CD123 to be expressed in theimmunophenotypically-defined leukemic stem cell (LSC) in ALL, raisingthe possibility that targeting CD123 on LSC could promote ALLeradication.

To study the role of CART 123 in antigen-loss relapses a novel xenograftmodel of CD19-negative relapses was developed from primary blastsderived from a B-ALL patient (CHP101) enrolled in one of the CTL019trials of the University of Pennsylvania/Children's Hospital ofPhiladelphia. This patient, at baseline had a classic CD19+CD123+phenotype but then relapsed after CART19 treatment with CD19−CD123+disease. Using this model, it was demonstrated CART123 could eradicatethe relapsed disease, and in combination with CART19 could preventantigen-loss relapse. This is the first demonstration of a dual CARTcombination in a clinically relevant, patient-derived model. Inaddition, through use of intravital imaging, it was shown that CARTcells enter the marrow in less than 24 hours after intravenousinjection, search for their targets, and slowdown in order to interactwith cognate antigen-bearing cells. Furthermore, it was shown that adual signaling CART123/19 was more effective than either CART alone or apool of both CAR, consistent with previously published results.

Previously, it was shown the pre-clinical efficacy of anti-CD123chimeric antigen receptor for the treatment of acute myeloid leukemia.CART123 causes hematopoietic toxicity due to recognition of CD123 onhematopoietic stem and progenitor cells, a potentially major challengeto clinical translation as profound stem cell toxicity could lead topermanent myeloablation. It was hypothesized that to minimizehematopoietic toxicity, a novel construct that activated T cells onlyupon co-engagement of CD19 and CD123 simultaneously could obviate thishematopoietic toxicity. Here it was found that CART cells receiving anactivating signal from CD19 recognition and a stimulatory signal fromCD123 recognition could kill B-ALL cells as well as avoid the profoundhematopoietic toxicity that we have previously described. Although thisconcept was previously published using an artificial system of CD19/PSMArecognition, this clinically relevant construct represents a majoradvance in the field with a relatively clear path to clinicaltranslation. Notably, although such a dual CAR design will likely beassociated with reduced toxicity, it may leave unaddressed the issue ofantigen loss relapse by making CAR recognition more, rather than lessrestrictive. However, if targeting of CD123 successfully eradicates ALLstem cells, this approach could be both safe and efficacious.

In summary, demonstrated here is a novel and effective strategy to thetreatment of B-ALL by targeting CD123. This approach is particularlyattractive since CD123 is expressed in rare CD19-negative malignantcells in some patients with B-ALL and is retained in antigen-lossrelapses occurring after CD19-directed immunotherapies. Moreover, thecombination of CART19 with CART123 can prevent the occurrence ofCD19-negative relapses.

Example 8 Enhancing CART Function and Therapeutic Index in Combinationwith Checkpoint Inhibitors

Chimeric antigen receptor T cell (CART) therapy has developed as apowerful and potentially curative therapy in hematological malignanciesover the last few years. CD19 directed CART cells have resulted inimpressive complete response rates of ˜90% in acute lymphoblasticleukemia that are durable for the majority of patients. However, theoverall response rates in other malignancies such as chronic lymphocyticleukemia are around 50%. This could be partially related to CARTexhaustion and dysfunction induced by leukemia cells. In this example,the role of inhibitory receptors/pathways were evaluated in inducingCART cell dysfunction and exhaustion in hematological malignancies.

As a tumor model, the acute myeloid leukemia (AML) cell line (MOLM14)and primary AML samples were treated with CD33 or CD123 directed CARTcells (comprising 41BB and CD3z stimulatory domains and a lentiviralvector, 1172 construct (SEQ ID NO: 707)).

Incubation of primary AML samples or MOLM14 cell line with CD33 or CD123directed CARTs resulted in a significant up-regulation of PDL1 on tumorcells after 24 hours of incubation (0% on day 0 vs 80% on day 1,P<0.001), and up-regulation of PD1 and TIM3 on T cells 3-7 days postco-culture (8% of T cells expressed PD1 on day 0 vs 43% on day 3, P=0.03and 13% of T cells expressed TIM3 on day 0 vs 71% on day 3, p=0.001,FIGS. 41A, 41B, and 41C). Further flow cytometry analysis demonstratedupregulation of checkpoint inhibitors PD1, TIM3, and additionalcheckpoint inhibitors LAG3 and CTLA4, in CD8+ T cells (FIGS. 42A, 42B,42C, and 42D) and in CD4+ T cells. Importantly, maximal expression wasobserved between days 3-7 after exposure to the target cells (primaryAML or MOLM14 cells).

For in-vivo experiments, NSG (NOD-SCID-g^(−/−)) mice were engrafted withMOLM14 cell line. Treatment of these AML xenografts with suboptimaldoses of CD33 or CD123 CARTs resulted in initial anti-tumor responses,followed by disease relapses in 40-60% of the mice (FIG. 44).

T cells were isolated from the bone marrow of these mice and analyzedfor differential expression of inhibitory receptors. There wassignificant increased up-regulation of PD1 and TIM-3 receptors on Tcells isolated from mice with relapsed disease compared T cells isolatedfrom mice in remission after CART cell therapy (FIGS. 43 and 45).

Next, the role of adding checkpoint inhibitors to improve T cellfunctions ex-vivo after CART cell therapy was investigated. T cellsisolated from bone marrows of mice that relapsed after CART cell therapywere co-cultured, in the presence of tumor, with a PD-1 inhibitor(BioXcell, Catalog No. is BE0193 and clone# is J110), a TIM3 inhibitor(Biolegend, clone F38-2E2, Catalog No. 345010), a CEACAM inhibitor(Sigma-Aldrich, Catalog No. SAB1403604-100UG), or the combination of PD1and TIM3 inhibitors, or the combination of PD1 and CEACAM1 inhibitors.The checkpoint inhibitors were administered at the concentration of 10ug/ml. There was an improvement of CART cells effector functions asmeasured by cytokine production (e.g., IFN-gamma) and Ki-67proliferation marker in the presence of checkpoint inhibitors. Theimprovement of the CART cell effector function was more pronounced whenboth PD1 and TIM3 inhibitors were combined (FIGS. 46A and 46B).

Finally, it was tested whether combining checkpoint inhibitors withCARTs would improve anti-tumor activity, e.g., augment the therapeuticindex and prevent relapses in AML xenografts. In this approach, MOLM14xenografts were treated with suboptimal doses of CD33 or CD123 directedCARTs or with control untransduced T cells (UTD), with or withoutdifferent combinations of checkpoint inhibitors. NSG mice were engraftedwith MOLM14 AML cell line. Engraftment was confirmed by bioluminescentimaging. The AML xenografts were then treated with suboptimal doses(0.25-0.5×10⁶ total T cells I.V) of CD33 or CD123 directed CARTs or withcontrol untransduced T cells (UTD). Mice also received PD-1 blockade,TIM3 blockade or the combination of both on days 3, 6, 9 and 12 post Tcells. Mice were followed with serial imaging to assess disease burden.The experimental schema is summarized in FIGS. 47 and 49.

The addition of checkpoint inhibitors to untransduced T cells did notlead to an anti-leukemic effect (FIG. 48). However, the addition of PD1or TIM3 blockade resulted in synergistic anti-tumor activity as shown inFIGS. 50A, 50B, 50C and 50D. The durable complete response rate was: 45%for treatment with CART123 alone (FIGS. 50A, 50B, and 50C), 80% fortreatment with CART123+PD1 inhibition (FIG. 50A), 100% for treatmentwith CART123+TIM3 inhibition (FIG. 50B), and 80% for treatment withCART123+ both PD1 and TIM3 inhibition (FIG. 50C).

Thus, treatment with CART123 in combination with checkpoint inhibitorsresulted in greater anti-tumor activity than the anti-tumor effectobserved when administering CART123 combined with the anti-tumor effectobserved when administering any of the checkpoint inhibitors alone.These results indicate that PD1 and TIM3 pathways are involved in CARTsexhaustion and dysfunction in AML. Combination of checkpoint inhibitorswith CART cells can lead to enhanced functions in AML and otherhematological malignancies.

Example 9 Low Dose RAD001 Stimulates CART Proliferation in a CellCulture Model

The effect of low doses of RAD001 on CAR T cell proliferation in vitrowas evaluated by co-culturing CART-expressing cells with target cells inthe presence of different concentrations of RAD001.

Materials and Methods

Generation of CAR-transduced T Cells

A humanized, anti-human CD19 CAR (huCART19) lentiviral transfer vectorwas used to produce the genomic material packaged into VSVg pseudotypedlentiviral particles. The amino acid and nucleotide sequence of thehumanized anti-human CD19 CAR (huCART19) is CAR 1, ID 104875 describedin PCT publication, WO2014/153270, filed Mar. 15, 2014, and isdesignated SEQ ID NOs. 85 and 31 therein.

Lentiviral transfer vector DNA is mixed with the three packagingcomponents VSVg env, gag/pol and rev in combination with lipofectaminereagent to transfect Lenti-X 293T cells. Medium is changed after 24 hand 30 h thereafter, the virus-containing media is collected, filteredand stored at −80° C. CARTs are generated by transduction of fresh orfrozen naïve T cells obtained by negative magnetic selection of healthydonor blood or leukopak. T cells are activated by incubation withanti-CD3/anti-CD28 beads for 24 h, after which viral supernatant orconcentrated virus (MOI=2 or 10, respectively) is added to the cultures.The modified T cells are allowed to expand for about 10 days. Thepercentage of cells transduced (expressing the CARs on the cell surface)and the level of CAR expression (relative fluorescence intensity, GeoMean) are determined by flow cytometric analysis between days 7 and 9.The combination of slowing growth rate and T cell size approaching ˜350fL determines the state for T cells to be cryopreserved for lateranalysis.

Evaluating Proliferation of CARTs

To evaluate the functionality of CARTs, the T cells are thawed andcounted, and viability is assessed by Cellometer. The number ofCAR-positive cells in each culture is normalized using non-transduced Tcells (UTD). The impact of RAD001 on CARTs was tested in titrations withRAD001, starting at 50 nM. The target cell line used in all co-cultureexperiments is Nalm-6, a human pre-B cell acute lymphoblastic leukemia(ALL) cell line expressing CD19 and transduced to express luciferase.

For measuring the proliferation of CARTs, T cells are cultured withtarget cells at a ratio of 1:1. The assay is run for 4 days, when cellsare stained for CD3, CD4, CD8 and CAR expression. The number of T cellsis assessed by flow cytometry using counting beads as reference.

Results

The proliferative capacity of CART cells was tested in a 4 dayco-culture assay. The number of CAR-positive CD3-positive T cells (darkbars) and total CD3-positive T cells (light bars) was assessed afterculturing the CAR-transduced and non-transduced T cells with Nalm-6(FIG. 53). huCART19 cells expanded when cultured in the presence of lessthan 0.016 nM of RAD001, and to a lesser extent at higher concentrationsof the compound. Importantly, both at 0.0032 and 0.016 nM RAD001 theproliferation was higher than observed without the addition of RAD001.The non-transduced T cells (UTD) did not show detectable expansion.

Example 10 Low Dose RAD001 Stimulates CART Expansion In Vivo

This example evaluates the ability of huCAR19 cells to proliferate invivo with different concentrations of RAD001.

Materials and Methods:

NALM6-luc cells: The NALM6 human acute lymphoblastic leukemia (ALL) cellline was developed from the peripheral blood of a patient with relapsedALL. The cells were then tagged with firefly luciferase. Thesesuspension cells grow in RPMI supplemented with 10% heat inactivatedfetal bovine serum.

Mice: 6 week old NSG (NOD.Cg-Prkdc^(scid)Il2rg^(tm1wjl)/Szb mice werereceived from the Jackson Laboratory (stock number 005557).

Tumor implantation: NALM6-luc cells were grown and expanded in vitro inRPMI supplemented with 10% heat inactivated fetal bovine serum. Thecells were then transferred to a 15 ml conical tube and washed twicewith cold sterile PBS. NALM6-luc cells were then counted and resuspendedat a concentration of 10×10⁶ cells per milliliter of PBS. The cells wereplaced on ice and immediately (within one hour) implanted in the mice.NALM6-luc cells were injected intravenously via the tail vein in a 100μl volume, for a total of 1×10⁶ cells per mouse.

CAR T cell dosing: Mice were administered 5×10⁶ CAR T cells 7 days aftertumor implantation. Cells were partially thawed in a 37 degree Celsiuswater bath and then completely thawed by the addition of 1 ml of coldsterile PBS to the tube containing the cells. The thawed cells weretransferred to a 15 ml falcon tube and adjusted to a final volume of 10mls with PBS. The cells were washed twice at 1000 rpm for 10 minuteseach time and then counted on a hemocytometer. T cells were thenresuspended at a concentration of 50×10⁶ CAR T cells per ml of cold PBSand kept on ice until the mice were dosed. The mice were injectedintravenously via the tail vein with 100 μl of the CAR T cells for adose of 5×10⁶ CAR T cells per mouse. Eight mice per group were treatedeither with 100 μl of PBS alone (PBS), or humanized CD19 CAR T cells.

RAD001 dosing: A concentrated micro-emulsion of 50 mg equal to 1 mgRAD001 was formulated and then resuspended in D5W (dextrose 5% in water)at the time of dosing. Mice were orally dosed daily (via oral gavage)with 200 μl of the desired doses of RAD001.

PK analysis: Mice were dosed daily with RAD001 starting 7 days posttumor implantation. Dosing groups were as follows: 0.3 mg/kg, 1 mg/kg, 3mg/kg, and 10 mg/kg. Mice were bled on days 0 and 14 following the firstand last dose of RAD001, at the following time points for PK analysis:15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and24 hours.

Results:

The expansion and pharmacokinetics of RAD001 was tested in NSG mice withNALM6-luc tumors. Daily oral dosing of RAD001 alone did not have animpact on the growth of NALM6-luc tumors (FIG. 54). The pharmacokineticanalysis of RAD001 shows that it is fairly stable in the blood of tumorbearing mice (FIGS. 55A and 55B). Both the day 0 and day 14 PK analysesshow that the RAD001 concentrations in the blood is above 10 nm even 24hours after dosing at the lowest dose tested (0.3 mg/kg).

Based on these doses, huCAR19 CAR T cells were dosed with and withoutRAD001 to determine the proliferative ability of these cells. Thehighest dose used was 3 mg/kg based on the levels of RAD001 in the blood24 hours after dosing. As the concentration of RAD001 was above 10 nM 24hours after the final dose of RAD001, several lower doses of RAD001 wereused in the in vivo study with CAR T cells. The CAR T cells were dosedIV one day prior to the start of the daily oral RAD001 dosing. Mice weremonitored via FACS for T cell expansion.

The lowest doses of RAD001 show an enhanced proliferation of the CAR Tcells (FIGS. 56A-56B). This enhanced proliferation is more evident andprolonged with the CD4⁺ CAR T cells than the CD8⁺ CAR T cells. However,with the CD8⁺ CAR T cells, enhanced proliferation can be seen at earlytime points following the CAR T cell dose. In embodiments, a RNA CARTcell can also be used in combination with checkpoint inhibitors.

Example 11 In Vitro Characterization of CART123 Termination Strategy

Termination strategies are of particular interest for minimizingtoxicity of CART123 therapy. One strategy for reducing CART123 activityinvolves the ablation of CD123 CAR-expressing T cells by co-expressingCAR123 with CD20, and using anti-CD20 antibody rituximab to target theCART123 cells for destruction. In this example, a CART123 cell isgenerated that co-expresses CD20, and characterized by various in vitroassays, such as degranulation and cytokine production capacity.

An expression vector for expressing a CD123 CAR and CD20 wasconstructed. The map of the vector is shown in FIG. 57. NVS2 CAR123(also referred to herein as CAR123-2) is composed of a humanized singlechain variable fragment directed against CD123, CD8 hinge, CD8transmembrane domain, 41BB and CD3 stimulatory domains. NVS2 CAR123 isunder EF1alpha promoter and is operably linked to the full CD20 moleculewith a P2A protein. Cells expressing CAR123 only are referred to hereinas CART123 cells, and cells co-expressing CAR123 and CD20 are referredto herein as CART123P2ACD20 cells.

For the CD107 degranulation assay, CART123 and CART123P2A CD20 cellswere cultured alone, with the CD123 positive cell line MOLM14, the CD123negative control cell line Jurkat, and with PMA/Ionomycin as a positivecontrol, in the presence of CD28, CD49d costimulatory molecules andmonensin. CD107a was measured by flow cytometry after 4 hours ofincubation. CART123 P2A CD20 cells undergo robust specific CD107adegranulation in response to CD123 positive target, similar to CART123cells (FIG. 58), thereby demonstrating that the introduction of CD20molecule does not adversely impact the degranulation of CART123 cells.

Cytokine production of the CART123P2A CD20 cells was also assessed.CART123 and CART123P2A CD20 cells were cultured alone, with the CD123positive cell line MOLM14, the CD123 negative control cell line Jurkat,and with PMA/Ionomycin as a positive control, in the presence of CD28,CD49d costimulatory molecules and monensin. The cells were harvestedafter four hours, fixed and permeabilized, stained for cytokines GM-CSF,TNFα, IFNγ, or IL-2, and flow cytometric analyses were performed.Results from these assays demonstrated that the majority of CART123 P2ACD20 cells produce GM-CSF (FIG. 59A), TNFα (FIG. 59B), IFNγ (FIG. 59C),and IL-2 (FIG. 59D) in response to CD123 positive target, similar toCART123 cells.

In a cytotoxicity assay, CART123 and CART123P2A CD20 cells wereincubated with MOLM14-luc for 24 h at different E:T ratios as indicated,and bioluminescence imaging was then performed as a measure of residualliving cells. The results showed that CART123 P2A CD20 cells resultspecific killing of the CD123 positive target MOLM14, that is comparableto CART123 cells (FIG. 60).

Rituximab-mediated cytotoxicity was next assessed for the CART123 P2ACD20 cells. CART123 and CART123 P2A CD20 were incubated with Rituximabat various concentrations, 0 μg/ml, 1 μg/ml, 10 μg/ml and 100 μg/ml.Cells were harvested at 0, 4, 12, and 48 hours, and stained for CD3. Thepercentage of T cell depletion was computed. Rituximab led to directcytotoxicity, at the concentration of 100 ug/ml and after 48 hours ofincubation in CART123P2A CD20 cells (FIG. 61B) but not in the CART123cells (FIG. 61A).

The mechanism by which the CART123P2ACD20 cell depletion is mediated wasinvestigated. CART123 P2A CD20 cells were incubated with rituximab atdifferent concentrations 0 μg/ml, 1 μg/ml, 10 μg/ml and 100 μg/ml, inthe presence of 15% rabbit complement. As shown in FIG. 62, rituximab 1μg/ml or 10 μg/ml resulted in depletion of the majority of CART123 P2ACD20 cells after 12 hours of incubation. Rituximab 100 ug/ml resulted indepletion of the majority of CART123 P2A CD20 cells after 4 hours ofincubation. CART123 P2A CD20 cell depletion is mediated by complementdependent cytotoxicity.

Example 12 Efficient Termination Strategies of CD123 CARTs

Many children and adults with acute myeloid leukemia (AML) relapse orare incurable with current treatment modalities, highlighting a need foralternative therapies. Chimeric antigen receptor T cells targeting CD123(CART123) have demonstrated potent anti-leukemia activity in murinexenograft models of human AML. However, CART123 treatment of miceengrafted with normal human hematopoietic cells resulted in profoundmyeloablation, raising concerns for severe hematologic toxicity inpatients with AML who may be treated with such therapies. Here, it wasevaluated whether T cell deletion after CART123-induced eradication ofAML could minimize this bystander toxicity without impairing leukemiacontrol, and thereby increase the therapeutic window of anti-AML CAR Tcell immunotherapy.

Methods

Three termination strategies in human AML xenograft models wereexamined: (1) T cell ablation with the anti-CD52 antibody alemtuzumabafter treatment with 1×10⁵-1×10⁶ T cells lentivirally-transduced withanti-CD123-41BB-CD3zeta CART123, (2) T cell ablation with the anti-CD20antibody rituximab after treatment with 1×10⁵-1×10⁶ CART123 engineeredto co-express CD20 (CART123/CD20), and (3) treatment with“biodegradable” anti-CD123 mRNA-electroporated CAR T cells(RNA-CART123). Mice engrafted with luciferase-expressing human AML celllines (MOLM14, MOLM13, U937) or primary AML specimens (n=3) were treatedwith CD123-redirected CAR T cells as above. The CD123 CAR constructutilized for this experiment is the 1172 construct (SEQ ID NO: 707), andwere administered to the mice at week 1. For T cell depletion studies,alemtuzumab 1 or 5 mg/kg was injected intraperitoneally (IP) at week 2,week 3, or week 4 (e.g., 1, 2, or 3 weeks post-CART123) to determineoptimal dosing and timing of T cell ablation. In subsequent studies,rituximab 10 mg/kg was injected IP 4 weeks after CART123/CD20, or 1×10⁷RNA-CART123 was injected intravenously at 5, 9, and 16 days after AMLengraftment. Mice were followed by weekly bioluminescent imaging and/orquantitative flow cytometry analyses of blood, spleen, and/or bonemarrow.

Results

CART123 treatment of CD123+AML xenografts induced marked T cellexpansion and leukemia eradication in vivo, resulting in long-termanimal survival (p<0.0001 vs untransduced T cell-treated controls).Minimal xenogeneic graft-versus-host effects were observed.

The results from serial ablation of CART123 in the xenograft model byalemtuzumab administration are shown in FIG. 72A. Quantification ofCART123 cells was also performed at the corresponding timepoints (FIG.72B) and show that the single doses of alemtuzumab rapidly eliminatedCART123 cells. One dose of alemtuzumab rapidly eliminated T cells in alltested models with best efficacy of 5 mg/kg dosing at week 4 (e.g., 3weeks post-CART123). Analysis of overall survival is shown in FIG. 72C,demonstrating that mice receiving alemtuzumab at week 3 or week 4 showedimproved survival compared to the mice receiving alemtuzumab at week 2.

CART123/CD20 eradicated AML with similar kinetics to those of CART123,and 1 dose of rituximab at 4 weeks post-CART123/CD20 infusion rapidlyeliminated T cells while preserving leukemia remission. Mice withCART123- or CART123/CD20-induced AML remission at time of alemtuzumab orrituximab administration remained leukemia-free for >12 weeks (FIG.72A), and animal survival did not differ from that of CD123-redirectedCAR T cell-treated mice that did not undergo T cell depletion (p=1.00).Conversely, CART123-treated mice with residual AML at time of earlieralemtuzumab administration experienced rapid AML progression, consistentwith effective prior T cell elimination (FIG. 72A).

Furthermore, AML rechallenge (“relapse”) of animals with previouslyalemtuzumab- or rituximab-ablated T cells resulted in rapid AMLproliferation without T cell re-emergence, confirming the completenessof T cell depletion. Non-ablated mice demonstrated CAR T cellre-expansion with rejection of CD123+ rechallenge (p<0.0001) (FIG. 72A).

Alemtuzumab treatment was also examined in a second in vivo model, apediatric patient-derived xenograft model. In this model, mice wereinjected with AML290 cells. Six weeks after AML engraftment, the micewere administered saline, untransduced T cells, or CART123 cells(represented at week 0). Alemtuzumab was administered at 5 mg/kg to asubset of the mice receiving CART123 cells at week 4. Alemtuzumabtreatment was shown to completely eliminate CART123 T cells in theperipheral blood (FIG. 73B). Analysis of tumor progression showed thatmice receiving CART123 demonstrated remission up to 8 weeks afterCART123 treatment. Mice that received treatment of alemtuzumab andablation of CART123 cells at week 4 remained in remission (FIG. 73A).Analysis of AML present in different organs of the xenografts afterCART123 dosing was determined by flow cytometry. Treatment with CART123cells resulted in ablation of AML cells in both the spleen (FIG. 74A)and bone marrow (FIG. 74B). Treatment with alemtuzumab 4 weeks afterCART123 dosing preserved remission, as demonstrated by sustainedablation of AML cells in the spleen (FIG. 74A) and bone marrow (FIG.74B).

RNA-CART123 most rapidly eliminated AML and facilitated long-term animalsurvival, although RNA-CART123 had expectedly shorter persistence invivo than did CART123 or CART123/CD20. Alemtuzumab or rituximab alonedid not inhibit AML proliferation in non-CART123-treated xenograftmodels vs AML-only controls (p=1.00).

Comparison of the three termination approaches described in this exampleis shown in FIG. 75.

Conclusions:

Alemtuzumab and rituximab completely eliminated CD123-redirected CAR Tcells in human AML xenograft models. Sustained leukemia remissionrequired CART123 or CART123/CD20 persistence for 4 weeks prior to T celltermination via alemtuzumab 5 mg/kg or rituximab 10 mg/kg post-CART123or post-CART123/CD20, respectively. Ongoing studies are investigatingefficacy of T cell elimination in additional primary AML xenograftmodels and against other anti-AML CAR T cell immunotherapies. These Tcell termination strategies may augment efficacy of CAR T cell therapyin patients with AML, particularly prior to potential hematopoietic stemcell transplantation.

Example 13 CAR19 and CAR123 Combinations

Most of B-acute lymphoblastic leukemia (B-ALL) blasts co-express CD19and CD123. Targeting both antigens at the same time can lead toincreased anti-tumor activity and reduce the rate of relapse. Asdescribed in Example 8, the combination of CAR19 and CAR123 hasincreased therapeutic efficacy. In this example, different constructsfor administering the combination of CAR19 and CAR123 are investigated.

One strategy to administer the combination of CAR19 and CAR123 isthrough a dual CART, wherein a T cell expresses both the CAR19 andCAR123 constructs. A single lentiviral vector carrying the sequences ofboth CAR19 and CAR123 (instead of 2 separate vectors) was constructed tofacilitate the dual-CART production for clinical use (FIG. 63A). Thislentiviral construct includes both CAR19 and CAR123 linked through a P2Adomain under the EF1 promoter. T cells were transduced with thelentiviral construct, cultured for 6 days, and then were stained with aCD19 scFv-specific antibody and CD123-His peptide and anti-His-PEantibody, for flow cytometry analysis. Results show that T cellsexpressing both CAR19 and CAR123 were detected (FIG. 63B).

In another strategy, a split CAR strategy was utilized to express CAR19and CAR123. In this strategy, a vector was constructed where a CAR123comprising the costimulatory domain 4-1BB was linked through a P2Adomain to a CAR19 comprising the primary signaling domain CD3zeta (FIG.64A). Specifically, the CAR123 comprised a CD123 scFv, CD8 hinge and CD8transmembrane domain, and the 4-1BB domain. The CAR19 comprised a CD19scFv, CD8 hinge and CD8 transmembrane domain, and the CD3 zeta domain.In this strategy, only recognition of both CD19 and CD123 by therespective scFv portions of the CAR will result in activation of thesplit CART cell. T cells were transduced with the lentiviral construct,cultured for 6 days, and then were stained with a CD19 scFv-specificantibody and CD123-His peptide and anti-His-PE antibody, for flowcytometry analysis. Results show that T cells expressing both CAR19 andCAR123 were detected (FIG. 64B).

Example 14 CD123 CART Therapy in Histiocytic Disorders

CD123 CART therapy may be useful in histiocytic disorders, such as BPDCNor mast cell disorders. In this example, experiments were performed toassess CART123 function in the context of blastic plasmacytoid dendriticcell neoplasm (BPDCN) and mast cell disorders (such as systemicmastocytosis and mast cell leukemia).

BPDCN is a rare and aggressive hematologic neoplasm arising from theprecursors of pDC, classified as a histiocyte/dendritic cell neoplasm.All BPDCN express CD123. CD123 appears to be critical for BPDCN survivalas BPDCN blasts require IL-3 for successful ex vivo propagation. Despiteinitially high response rates to chemotherapy, long-term survival isusually very poor, and median survival is 9-13 months irrespective ofinitial presentation.

CD123 expression was determined by flow cytometry on hematopoietic stemcells (HSC), acute myeloid leukemia cell line (AML), or two differentBPDCN. Median fluorescence intensity is indicated. The expression ofCD123 on AML, normal marrow CD34+ cells, and two cases of BPDCN areshown in FIG. 65. These data indicate that CD123 is expressed at10-20-fold higher levels on BPDCN compared with normal marrow.

Additional CART123 constructs were generated using alternativeanti-CD123 clones (32716 (also referred to herein as the 1172 construct)and 26292 (also referred to herein as the 1176 construct), bothconstructs are described and characterized in PCT/US2014/017328. Cellkilling assays were performed similarly to those described in Example 2or 3. Clone 26292 led to reduced killing of normal CD34 cells comparedwith positive control AML or BPDCN, indicating that it is feasible toproduce CART-123 with attenuated activity that might generate anenhanced therapeutic window (FIG. 66B). In contrast, clone 32716 showedsimilar killing of CD34 cells and BPDCN (FIG. 66A).

Sensitive analyses of T cell function such as degranulation and cytokineproduction were performed similarly to those described in Example 2 or3. Clone 26292 CART123 were incubated with CD34+ HSC (FIG. 67, top row)or BPDCN (FIG. 67, bottom row) for four hours. CD107 degranulation wasassessed. Results from the degranulation assay showed that effectorfunctions of clone 26292 can still be triggered by CD34+HSC (FIG. 67,top row).

Mast cell disorders, including systemic mastocytosis and mast cellleukemia, are rare histiocytic malignancies that are associated with apoor prognosis. Mast cells express CD123 at high levels and thus mastcell disorders could be treated with CART123.

Analysis of CD123 expression on mast cell disorders was performed.Mononuclear cells from the blood of a patient with mast cellleukemia/systemic mastocytosis were stained with live/dead aqua (LDAQ, xaxis in FIGS. 68A and 68B) and isotype (FIG. 68A) or CD123 PE (FIG.68B). Most SM cells express CD123.

A CD107 degranulation assay was performed to assess CART123 activity inthe presence of mast cell leukemia cells. CART123 cells were culturedalone, with PMA and ionomycin (FIG. 69A) or with mononuclear cells fromthe blood of a patient with mast cell leukemia/systemic mastocytosis(FIG. 69B) for two hours, in the presence of anti-CD107a antibody. Tcells were gated using anti-CD3. CD107a positivity was gated using theCART123 alone tube. Response of CART123 to SM cells is equivalent tothat generated with the positive control PMA+ionomycin.

Example 15 Effect of CART123 on the Tumor Microenvironment

In this example, assays are performed to test the effect of CD123CAR-expressing cells on the tumor microenvironment. These assays can beused to assess the effect of CART123 on the tumor microenvironment inHodgkin's Lymphoma.

A first assay is performed to determine whether malignant cells, e.g.,Hodgkin Lymphoma cells (HL cells), lead to an immunosuppressivephenotype in cells in the tumor microenvironment, e.g., monocytes.Monocyte cells and HL cells are grown in a transwell plate comprising anupper and a lower chamber. The monocyte and malignant cells are culturedin the following configurations: (1) the monocyte cell is grown in onechamber, e.g., the lower chamber, and the HL cell is grown in the otherchamber, e.g., the upper chamber, (2) the monocyte cell and HL cell aregrown together in the same side of a transwell plate, or (3) themonocyte cell is grown alone (control). The cells are cultured for adesired period of time, and then the cells are harvested for flowcytometry analysis or real-time quantitative PCR to detect the monocytephenotype, e.g., immunosuppressive phenotype, by assessing the levels ofmarkers such as CD14 and HLADR.

A second assay is performed to determine whether the immunosuppressivetumor microenvironment cells, e.g., monocytes, can inhibit anti-tumor Tcell immunity mediated by CART cells. This assay can be performed in twodifferent ways, using a transwell plate if the inhibition of anti-tumorT cell immunity is suspected to be through a soluble factor, or usingstandard cell culture containers if the inhibition of anti-tumor T cellimmunity is suspected to be contact-mediated. For an assay whereinhibition of anti-tumor T cell immunity may be through a solublefactor, cells are grown in a transwell plate comprising an upper and alower chamber. The cells are cultured in the following configurations:(1) a HL cell in one chamber, e.g., upper chamber, and a CART19 cell anda CD19-expressing tumor cell, e.g., a B cell tumor, in the otherchamber, e.g., lower chamber; (2) a monocyte in one chamber, e.g., upperchamber, and a CART19 cell and a CD19-expressing tumor cell, e.g., a Bcell tumor, in the other chamber; and (3) a HL cell and a monocyte inone chamber, e.g., upper chamber, and a CART19 cell and aCD19-expressing tumor cell, e.g., a B cell tumor, in the other chamber.For an assay where inhibition of anti-tumor T cell immunity may becontact-mediated, the cells are cultured in the followingconfigurations: (1) a HL cell, a CART19 cell, and a CD19-expressingtumor cell; (2) a monocyte, a CART19 cell, and a CD19-expressing tumorcell; and (3) a HL cell, a monocyte, a CART19 cell, and aCD19-expressing tumor cell. The cells are cultured for a desired periodof time, and then CART19 function is assessed. Assays to assess CART19function include proliferation and killing assays, e.g., as described inExamples 2 and 3.

A third assay is performed to determine that CART123 targetsimmunosuppressive cells in the tumor microenvironment. This assay can beperformed in two different ways, using a transwell plate if theinhibition of anti-tumor T cell immunity is suspected to be through asoluble factor, or using standard cell culture containers if theinhibition of anti-tumor T cell immunity is suspected to becontact-mediated. For an assay where inhibition of anti-tumor T cellimmunity may be through a soluble factor, cells are grown in a transwellplate comprising an upper and a lower chamber. The cells are cultured inthe three transwell plate configurations for the second assay describedabove, and in addition, are cultured in the following configurationswhich include the CART123 cell: (4) a HL cell and CART123 cell in onechamber, e.g., upper chamber, and a CART19 cell and a CD19⁺CD123⁻ tumorcell, e.g., a B cell tumor, in the other chamber, e.g., lower chamber;(5) a monocyte and CART123 cell in one chamber, e.g., upper chamber, anda CART19 cell and a CD19⁺CD123⁻ tumor cell, e.g., a B cell tumor, in theother chamber; and (6) a HL cell, a monocyte, and a CART123 cell in onechamber, e.g., upper chamber, and a CART19 cell and a CD19⁺CD123⁻ tumorcell, e.g., a B cell tumor, in the other chamber. For an assay whereinhibition of anti-tumor T cell immunity may be contact-mediated, thecells are cultured in the three standard cell culture containers for thesecond assay described above, and in addition, are cultured in thefollowing configurations which include the CART123 cell: (4) a HL cell,a CART19 cell, a CART123 cell, and a CD19⁺CD123⁻ tumor cell; (5) amonocyte, a CART19 cell, a CART123 cell, and a CD19⁺CD123⁻ tumor cell;and (6) a HL cell, a monocyte, a CART19 cell, a CART123 cell, and aCD19⁺CD123⁻ tumor cell. The cells are cultured for a desired period oftime, and then CART19 function is assessed. Assays to assess CART19function include proliferation and killing assays, e.g., as described inExamples 2 and 3.

Example 16 Clinical Study of RNA CART123 in Refractory or Relapsed AML

This study addresses the feasibility, safety and efficacy ofintravenously administered, RNA electroporated autologous T cellsexpressing anti-CD123 chimeric antigen receptors expressing tandem TCRζand 4-1BB (TCRζ/4-1BB) costimulatory domains (referred to as “RNACART123”) in Acute Myeloid Leukemia (AML) subjects. This study evaluates15 subjects. Evaluable subjects are those who receive at least one doseof RNA CART123 cells. The duration of active protocol intervention isapproximately 2 months from screening visit. Subjects are followed for 6months after their first infusion.

The primary objectives of this study is to assess the safety of RNACART123 in AML subjects by recording the frequency and severity ofadverse events, including but not limited to, estimating the frequencyof CRS (cytokine release syndrome) and MAS (macrophage activationsyndrome). The secondary objectives of this study include: (1) determinepersistence and trafficking of RNA CART123 cells; (2) estimate theefficacy of at least 1 dose of RNA CART123 cells in AML subjects bymeasuring reduction of blast counts in the peripheral blood and marrow;(3) Estimate the efficacy of at least 1 dose of RNA CART123 cells in AMLsubjects by measuring the overall response rate (ORR) at 28+/−5 days,using (i) standard morphologic complete response criteria (malignantblasts <5% with count recovery), (ii) malignant blasts <5% without countrecovery, and (iii) minimal residual disease assessment; (4) determineoverall survival (OS) and progression-free survival (PFS) and cause(s)of death for all subjects until 6 months post RNA CART123 cell infusion;(5) determine the duration of response (DOR) for responding subjectsuntil 6 months post RNA CART123 cell infusion; and (6) determine thepercentage of subjects proceeding to allogeneic HCT (or secondallogeneic HCT).

Eligible subjects are male or female over 18 years old with AML ormyelodysplastic syndrome with no available curative treatment optionsusing currently available therapies. Subjects with second or subsequentrelapse, any relapse refractory to salvage, or with persistent diseaseafter at least two lines of therapy. Subjects must have evaluabledisease >5% blasts on marrow aspirate or biopsy, or extramedullarydisease (CNS involvement is prohibited) within 2 weeks prior toscreening.

Subjects are treated with IV administration of RNA anti-CD123 CAR Tcells for a total of up to six doses over 2 weeks. Dosing is accordingto subject weight with an in-subject dose escalation. Subjects aredivided into two cohorts. Cohort 1 includes the first 3 subjects toreceive RNA CART123 cells, and receives 3 escalating doses of RNACART123 cells, with no lymphodepleting chemotherapy prior to infusion(FIG. 70). Cohort 2 includes remaining 12 subjects of the study, andreceives up to six IV doses of RNA CART123 cells at an escalating dose(FIG. 71). Cohort 2 may be given lymphodepleting chemotherapy 4 days(+/−1 day) prior to the first CART123 cell infusion (if ALC>500/uL).Lymphodepleting chemotherapy may be repeated before the fourth dose ofRNA CART123 cells (if ALC>500/uL). Lymphodepleting chemotherapy includesa single dose of cyclophosphamide (1 mg/m²). The lymphodepletingchemotherapy dose within the RNA CART123 treatment regimen is designedto enhance engraftment of the subsequent T cell doses by enhancinghomeostatic space.

The doses of CART cells by weight are provided in the table below:

Subject Total weight Dose 1 Dose 2 Doses 3-6 CAR+ cells  <100 kg 1 ×10⁶/kg 2 × 10⁶/kg 4 × 10⁶/kg 1.9 × 10⁷/kg ≧100 kg 1 × 10⁸ 2 × 10⁸ 4 ×10⁸ 1.9 × 10⁹Weight used for dosing can be the weight obtained prior to the apheresisprocedure. Cell numbers are based on CAR+ cells with CAR expressiondetermined by flow cytometry. Dosing will not be changed for changes insubject weight. The indicated doses are +/−20% to account formanufacturing variability.

A sample RNA CART123 dosing schedule is provided below:

Study Day of mid- treatment Day 0 infusion Study Days ofcyclophosphamide occurs on: subsequent infusions: (Cohort 2), if givenMonday D 2, D 4, D 9*, D 11*, D 14* D 7 D 2, D 4, D 7*, D 9*, D 11* NoWednesday D 2, D 5, D 9*, D 12*, D 14* D 7 D 2, D 5, D 7*, D 9*, D 11*No Friday D 3, D 5, D 10*, D 12*, D 14* D 7 D 3, D 5, D 7*, D 10*, D 12*No *Infusions for subjects in Cohort 2 only

Baseline and subsequent tumor assessments will be performed according tostandard of care clinical practice for AML. Baseline AML marrowassessment can be performed if the subject's last marrow was performedmore than 2 weeks prior to the first cyclophosphamide dose. Subjectswill have marrow assessment for leukemia response at Day 16+/−2 (orwithin 7 days of the last RNA CART123 infusion received), Day 28+/−5,and at both 3 and 6 months (for subjects who did not proceed to analloHCT). CART cell levels and cytokines will be determined by theappropriate test at the TCSL research laboratory. Disease responsedefinitions can be as standard for AML.

All subjects with marrow aplasia at D28+/−5 (or 14 days after the last Tcell infusion, whichever occurs earlier) will undergo allogeneichematopoietic cell transplantation (alloHCT) as a rescue strategy. Allsubjects should therefore have a previously identified stem cell donor.Marrow aplasia is defined as >75% reduction in cellularity from normalfor age, in conjunction with ANC <0.5 k/ul or platelets <50 k/ul.

Treatment limiting toxicity (TLT) is defined as any unexpected eventthat is possibly, probably, or definitely related to the T cellinfusion, including non-hematological grade 3 or greater adverse eventand any Grade 3 or greater hypersensitivity reaction and autoimmunereaction as defined in Section 5.5. The following events do not compriseTLT: fever and infection, cytopenias of any grade, tumor lysis syndrome,cytokine release syndrome or any metabolic or laboratory abnormalitiesthat resolve to Grade 2 or lower within seven days.

Adverse event reporting begins at the start of the first dose of RNACART123 cells and will continue through Month 4 or until conditioningfor HCT or another alternative therapy is initiated, whichever occursearlier. Subjects can be continually reassessed for evidence of acuteand cumulative toxicity.

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

What is claimed is:
 1. An isolated nucleic acid molecule encoding achimeric antigen receptor (CAR), wherein the CAR comprises a CD123binding domain, a transmembrane domain, and an intracellular signalingdomain, and wherein said CD123 binding domain comprises: (a) a heavychain variable domain comprising three complementary determining regionsof heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) present in order of HCCDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acidsequence of SEQ ID NO: 487, HC CDR2 comprises the amino acid sequence ofSEQ ID NO: 492, and HC CDR3 comprises the amino acid sequence of SEQ IDNO: 497; and a light chain variable domain comprising threecomplementary determining regions of light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LCCDR1 comprises the amino acid sequence of SEQ ID NO: 502, LC CDR2comprises the amino acid sequence of SEQ ID NO: 507, and LC CDR3comprises the amino acid sequence of SEQ ID NO: 512; or (b) a heavychain variable domain comprising three complementary determining regionsof heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) present in order of HCCDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acidsequence of SEQ ID NO: 517, HC CDR2 comprises the amino acid sequence ofSEQ ID NO: 522, and HC CDR3 comprises the amino acid sequence of SEQ IDNO: 527; and a light chain variable domain comprising threecomplementary determining regions of light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LCCDR1 comprises the amino acid sequence of SEQ ID NO: 532, LC CDR2comprises the amino acid sequence of SEQ ID NO: 537, and LC CDR3comprises the amino acid sequence of SEQ ID NO: 542; or (c) a heavychain variable domain comprising three complementary determining regionsof heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) present in order of HCCDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acidsequence of SEQ ID NO: 335, HC CDR2 comprises the amino acid sequence ofSEQ ID NO: 363, and HC CDR3 comprises the amino acid sequence of SEQ IDNO: 391; and a light chain variable domain comprising threecomplementary determining regions of light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LCCDR1 comprises the amino acid sequence of SEQ ID NO: 419, LC CDR2comprises the amino acid sequence of SEQ ID NO: 447, and LC CDR3comprises the amino acid sequence of SEQ ID NO:
 475. 2. The isolatednucleic acid molecule of claim 1, which encodes a CAR comprising: (i)the amino acid sequence of the light chain variable region of SEQ ID NO:276; or (ii) an amino acid sequence with 95-99% identity to the aminoacid sequence of the light chain variable region of SEQ ID NO:
 276. 3.The isolated nucleic acid molecule of claim 1, which encodes a CARcomprising: (i) the amino acid sequence of the heavy chain variableregion of SEQ ID NO: 217; or (ii) an amino acid sequence with 95-99%identity to the amino acid sequence of the heavy chain variable regionof SEQ ID NO:
 217. 4. The isolated nucleic acid molecule of claim 1,which encodes a CAR comprising the amino acid sequence of the lightchain variable region of SEQ ID NO: 276, and the amino acid sequence ofthe heavy chain variable region of SEQ ID NO:
 217. 5. The isolatednucleic acid molecule of claim 1, wherein the encoded CD123 bindingdomain comprises: (i) the amino acid sequence of SEQ ID NO:480; (ii) anamino acid sequence having at least one, two or three modifications butnot more than 10 modifications to SEQ ID NO: 480; or (iii) an amino acidsequence with 95-99% identity to SEQ ID NO:
 480. 6. The isolated nucleicacid molecule of claim 1, wherein the CD123 binding domain comprises anucleotide sequence chosen from SEQ ID NO: 479 and 481, or a nucleotidesequence with 95-99% identity thereof.
 7. The isolated nucleic acidmolecule of claim 1, wherein: (i) the encoded CAR comprises atransmembrane domain that comprises a transmembrane domain of a proteinselected from the group consisting of the alpha, beta or zeta chain ofthe T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; or (ii) theencoded transmembrane domain comprises the amino acid sequence of SEQ IDNO: 6, or an amino acid sequence with 95-99% identity to the amino acidsequence of SEQ ID NO:6; or (iii) the nucleic acid sequence encoding thetransmembrane domain comprises nucleotides 928-999 of sequence of SEQ IDNO: 40, or a nucleotide sequence with 95-99% identity thereof.
 8. Theisolated nucleic acid molecule of claim 1, wherein the encoded CD123binding domain is connected to the transmembrane domain by a hingeregion, wherein (i) the encoded hinge region comprises the amino acidsequence of SEQ ID NO:2, or an amino acid sequence with 95-99% identityto SEQ ID NO: 2; or (ii) the nucleic acid sequence encoding the hingeregion comprises nucleotides 793-927 of sequence of SEQ ID NO: 40, or anucleotide sequence with 95-99% identity thereof.
 9. The isolatednucleic acid molecule of claim 1, wherein the encoded intracellulardomain comprises a costimulatory domain, wherein the costimulatorydomain comprises a functional signaling domain derived from a proteinselected from the group consisting of a MHC class I molecule, a TNFreceptor protein, an Immunoglobulin-like protein, a cytokine receptor,an integrin, a signaling lymphocytic activation molecule (SLAM protein),an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2,CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD18), 4-1BB(CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, and PAG/Cbp.
 10. The isolated nucleic acidmolecule of claim 9, wherein the costimulatory domain comprises theamino acid sequence of SEQ ID NO:7; or an amino acid sequence with95-99% identity to the amino acid sequence of SEQ ID NO:7.
 11. Theisolated nucleic acid molecule of claim 9, wherein the nucleic acidsequence encoding the costimulatory domain comprises nucleotides1000-1125 of sequence of SEQ ID NO: 40, or a nucleotide sequence with95-99% identity thereof.
 12. The isolated nucleic acid molecule of claim1, wherein the encoded intracellular signaling domain comprises afunctional signaling domain of 4-1BB and a functional signaling domainof CD3 zeta.
 13. The isolated nucleic acid molecule of claim 1, whereinthe encoded intracellular signaling domain comprises the amino acidsequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO:9 orSEQ ID NO:10; or an amino acid sequence with 95-99% identity to theamino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ IDNO:9 or SEQ ID NO:10.
 14. The isolated nucleic acid molecule of claim 1,wherein the encoded intracellular signaling domain comprises the aminoacid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9or SEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain.
 15. The isolated nucleic acid molecule of claim 1,wherein the nucleic acid sequence encoding the intracellular signalingdomain comprises nucleotides 1000-1125 of sequence of SEQ ID NO: 40, ora nucleotide sequence with 95-99% identity thereof, and/or nucleotides1126-1461 of SEQ ID NO: 40 or SEQ ID NO:21, or a nucleotide sequencewith 95-99% identity thereof.
 16. The isolated nucleic acid molecule ofclaim 1, further comprising a leader sequence which encodes the aminoacid sequence of SEQ ID NO:1.
 17. The isolated nucleic acid molecule ofclaim 1, which encodes a CAR comprising: (i) the amino acid sequence ofSEQ ID NO: 99; (ii) an amino acid sequence having at least one, two orthree modifications but not more than 10 modifications to SEQ ID NO: 99;or (iii) an amino acid sequence with 95-99% identity to SEQ ID NO: 99.18. The isolated nucleic acid molecule of claim 1, comprising thenucleotide sequence of SEQ ID NO: 40, or a nucleotide sequence with95-99% identity to SEQ ID NO:
 40. 19. An isolated chimeric antigenreceptor (CAR) polypeptide, wherein the CAR comprises a CD123 bindingdomain, a transmembrane domain, and an intracellular signaling domain,and wherein said CD123 binding domain comprises: (a) a heavy chainvariable domain comprising three complementary determining regions ofheavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) present in order of HCCDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acidsequence of SEQ ID NO: 487, HC CDR2 comprises the amino acid sequence ofSEQ ID NO: 492, and HC CDR3 comprises the amino acid sequence of SEQ IDNO: 497; and a light chain variable domain comprising threecomplementary determining regions of light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LCCDR1 comprises the amino acid sequence of SEQ ID NO: 502, LC CDR2comprises the amino acid sequence of SEQ ID NO: 507, and LC CDR3comprises the amino acid sequence of SEQ ID NO: 512; or (b) a heavychain variable domain comprising three complementary determining regionsof heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) present in order of HCCDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acidsequence of SEQ ID NO: 517, HC CDR2 comprises the amino acid sequence ofSEQ ID NO: 522, and HC CDR3 comprises the amino acid sequence of SEQ IDNO: 527; and a light chain variable domain comprising threecomplementary determining regions of light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LCCDR1 comprises the amino acid sequence of SEQ ID NO: 532, LCCDR2comprises the amino acid sequence of SEQ ID NO: 537, and LC CDR3comprises the amino acid sequence of SEQ ID NO: 542; or (c) a heavychain variable domain comprising three complementary determining regionsof heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) present in order of HCCDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acidsequence of SEQ ID NO: 335, HC CDR2 comprises the amino acid sequence ofSEQ ID NO: 363, and HC CDR3 comprises the amino acid sequence of SEQ IDNO: 391; and a light chain variable domain comprising threecomplementary determining regions of light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LCCDR1 comprises the amino acid sequence of SEQ ID NO: 419, LC CDR2comprises the amino acid sequence of SEQ ID NO: 447, and LC CDR3comprises the amino acid sequence of SEQ ID NO:
 475. 20. The isolatedCAR polypeptide of claim 19, comprising: (i) the amino acid sequence ofthe light chain variable region of SEQ ID NO: 276; or (ii) an amino acidsequence with 95-99% identity to the amino acid sequence of the lightchain variable region of SEQ ID NO:
 276. 21. The isolated CARpolypeptide of claim 19, comprising: (i) the amino acid sequence of theheavy chain variable region of SEQ ID NO: 217; or (ii) an amino acidsequence with 95-99% identity to the amino acid sequence of the heavychain variable region of SEQ ID NO:
 217. 22. The isolated CARpolypeptide of claim 19, comprising the amino acid sequence of the lightchain variable region of SEQ ID NO: 276, and the amino acid sequence ofthe heavy chain variable region of SEQ ID NO:
 217. 23. The isolated CARpolypeptide of claim 19, comprising: (i) the amino acid sequence of SEQID NO:480; (ii) an amino acid sequence having at least one, two or threemodifications but not more than 10 modifications to SEQ ID NO: 480; or(iii) an amino acid sequence with 95-99% identity to SEQ ID NO:
 480. 24.The isolated CAR polypeptide of claim 19, wherein the transmembranedomain comprises a transmembrane domain from a protein selected from thegroup consisting of the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
 25. The isolated CARpolypeptide of claim 19, wherein the transmembrane domain comprises: (i)the amino acid sequence of SEQ ID NO: 6, or (ii) a sequence with 95-99%identity to the amino acid sequence of SEQ ID NO:6.
 26. The isolated CARpolypeptide of claim 19, wherein the CD123 binding domain is connectedto the transmembrane domain by a hinge region, wherein the hinge regioncomprises SEQ ID NO:2, or a sequence with 95-99% identity thereof. 27.The isolated CAR polypeptide of claim 19, wherein the encodedintracellular domain comprises a costimulatory domain, wherein thecostimulatory domain comprises a functional signaling domain derivedfrom a protein selected from the group consisting of a MHC class Imolecule, a TNF receptor protein, an Immunoglobulin-like protein, acytokine receptor, an integrin, a signaling lymphocytic activationmolecule (SLAM protein), an activating NK cell receptor, BTLA, a Tollligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1,LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278),GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX,CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, and PAG/Cbp.
 28. The isolatedCAR polypeptide of claim 19, wherein the costimulatory domain comprisesthe amino acid sequence of SEQ ID NO:7, or a sequence with 95-99%identity to the amino acid sequence of SEQ ID NO:7.
 29. The isolated CARpolypeptide of claim 19, wherein the intracellular signaling domaincomprises a functional signaling domain of 4-1BB and a functionalsignaling domain of CD3 zeta.
 30. The isolated CAR polypeptide of claim19, wherein the intracellular signaling domain comprises the amino acidsequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO:9 orSEQ ID NO:10; or an amino acid sequence with 95-99% identity to theamino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ IDNO:9 or SEQ ID NO:10.
 31. The isolated CAR polypeptide of claim 19,comprising: (i) the amino acid sequence of SEQ ID NO: 99; (ii) an aminoacid sequence having at least one, two or three modifications but notmore than 10 modifications to SEQ ID NO: 99; or (iii) an amino acidsequence with 95-99% identity to SEQ ID NO:
 99. 32. A vector comprisingthe nucleic acid molecule of claim 1, wherein the vector is a DNAvector, an RNA vector, a plasmid, a lentivirus vector, adenoviralvector, or a retrovirus vector.
 33. An isolated immune effector cellcomprising the nucleic acid molecule of claim
 1. 34. A method of makingan immune effector cell, comprising transducing the immune effector cellwith the vector of claim
 32. 35. A method of generating a population ofRNA-engineered cells, comprising introducing an in vitro transcribed RNAor synthetic RNA into a cell, wherein the RNA comprises the nucleic acidmolecule of claim
 1. 36. The isolated cell of claim 33, furtherexpressing a chimeric molecule that comprises a first polypeptide thatcomprises at least a portion of an inhibitory molecule, associated witha second polypeptide that comprises a positive signal from anintracellular signaling domain.
 37. The isolated nucleic acid moleculeof claim 1, wherein the encoded intracellular signaling domain comprisesa primary signaling domain comprising a functional signaling domainderived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3delta , CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10,DAP12, or CD66d.
 38. The isolated nucleic acid molecule of claim 37,wherein the primary signaling domain comprises a functional signalingdomain of CD3 zeta, wherein the CD3 zeta comprises the amino acidsequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence with 95-99%identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.39. The isolated CAR polypeptide of claim 19, wherein the intracellularsignaling domain comprises a primary signaling domain comprising afunctional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma,FcR beta, CD3 gamma, CD3 delta , CD3 epsilon, CD5, CD22, CD79a, CD79b,CD278, FcεRI, DAP10, DAP12, or CD66d.
 40. The isolated CAR polypeptideof claim 39, wherein the primary signaling domain comprises a functionalsignaling domain of CD3 zeta, wherein the CD3 zeta comprises the aminoacid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence with95-99% identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:10.